Chromatographic isolation of cells and other complex biological materials

ABSTRACT

The present invention relates to the chromatographic isolation of a target cell or another complex biological material, in particular by column chromatography such as affinity chromatography or gel permeation chromatography. The invention employs a receptor binding reagent that binds to a receptor molecule that is located on the surface of a target cell. The invention in general provides novel methods for the traceless isolation of biologic materials such as cells, cell organelles, viruses and the like. The invention also relates to an apparatus for the isolation of cells and other complex biological materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/380,699, filed Aug. 22, 2014, entitled “Chromatographic Isolation OfCells And Other Complex Biological Materials,” which claims the benefitof priority to International Application No. PCT/EP2013/053650, filedFeb. 25, 2013, entitled “Chromatographic Isolation Of Cells And OtherComplex Biological Materials,” and U.S. provisional application No.61/602,150 filed Feb. 23, 2012, entitled “Chromatographic Isolation OfCells And Other Complex Biological Materials,” the contents of which areincorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled735042009501SeqList.txt, created Dec. 20, 2018, which is 6,730 bytes insize. The information in electronic format of the Sequence Listing isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the chromatographic isolation of atarget cell or a different (complex) biological material, in particularby column chromatography such as affinity chromatography or gelpermeations chromatography. The invention employs a receptor bindingreagent that binds to a receptor molecule that is located on the surfaceof a target cell. The method discloses herein can also be described as(traceless) cell affinity chromatography technology (CATCH). Theinvention in general provides novel methods for the traceless isolationof biologic materials such as cells, cell organelles, viruses and thelike. The invention also relates to an apparatus for the isolation ofcells and other complex biological materials.

BACKGROUND OF THE INVENTION

Isolation of pure and functional cell populations of a desired cell typeis a prerequisite in a variety of therapeutic, diagnostic, andbiotechnological applications.

Bonnafous et al., J. Immunol. Methods. 1983 Mar. 11; 58 (1-2):93-107describe a cell affinity chromatography with ligands immobilized throughcleavable mercury-sulfur bonds, that means ligands that are immobilizedvia covalent bonds. In this method, Bonnafous et al conjugate theorganomercurial mersalyl to trisacryl beads bearing primary aminogroups. According to Bonnafous et al, thiolated ligands can becovalently immobilized on this matrix through cleavable Hg—S bonds. Twomodel studies of cell separation are reported by Bonnafous et al: (i)concanavalin A thiolated withN-succinimidyl-3-(2-pyridyldithio)-propionate and immobilized onmersalyl-trisacryl; mouse thymocytes bound to Con A-mersalyl-trisacrylwere eluted from the support by short thiol treatment which preservedcell viability; (ii) anti-dinitrophenyl antibodies modified withS-acetyl-mercaptosuccinic anhydride and immobilized onmersalyl-trisacryl; sheep erythrocytes, previously labelled withtrinitrobenzene sulfonic acid, bound to this support and were recoveredby thiol treatment without hemolysis.

In this context it is noted that chromatography is a well-establishedtechnique for the separation of low molecular weight and high molecularweight molecules, including proteins. This technique has also beenapplied to cell separation, in particular in the form of affinitychromatography using immobilized ligands specific to a desired celltype, such as immunoligands. As an example, different T cell subsetshave been separated by labelling with monoclonal immunoglobulins andloading onto a column with polyacrylamide beads, to which rabbitanti-mouse lgG was covalently bound (Braun, R., et al., Journal ofImmunological Methods (1982) 54, 251-258). As a further example,lectin-affinity column chromatography, using Sepharose 6 MB covalentlyconjugated to Dolichos biflorus agglutinin, has been used to separateleukemic cells from healthy leukocytes (Ohba, H., et al, Cancer Letters(2002) 184, 207-214).

As cells are generally by magnitudes larger than proteins they hardlyenter, in contrast to proteins, the pores of the beads of conventionalchromatography sorbents. Using sorbents with large pores does notsignificantly overcome this separation phenomenon due to diffusionallimitations. On the other hand, the surface area within pores onlyaccessible for proteins usually largely exceeds the surface areaaccessible for both proteins and cells. Therefore, the use ofconventional chromatography sorbents for the immobilization ofproteinaceous or other receptor binding ligands for the generation of anaffinity matrix for cells usually requires the use of a wasteful largeexcess of receptor binding ligands as most of them are immobilized inpores or cavities that cannot be accessed by the cells. Specificreceptor binding reagents are often expensive and difficult to beproduced at the desired scales thereby bringing this aspect to seriousconsideration. The use of monolithic sorbents in the form of cryogelshas therefore been suggested as an alternative technique in affinitychromatography of cells (see e.g. Dainiak, M. B., et al., Adv. Biochem.Engin./Biotechnol. (2007), 106, 101-127). However, monolithic sorbentsare scarce so that a desired sorbent may not be commercially availablein the form of a monolithic column. Furthermore, in case of affinitychromatography, generally the need remains to remove a competingcompound used to elute the desired cells from these cells. Potentialadvantages of monolithic sorbents in terms of cell viability may thus bereversed by additional procedures required to remove the compound usedto elute the cells from the affinity chromatography column.

The most important currently used cell isolation methods aremagnet-assisted cell sorting (MACS) and fluorescence-assisted cellsorting (FACS™). Cell sorting by flow cytometry, where typicallyfluorophores, coupled to antibodies, are used to label cells, analysescells individually. Cells are separated at high speed under very highpressures using a cell sorting apparatus. FACS™ technology enablesisolation of cells defined by a set of markers in one step by applying acorresponding set of antibodies with different fluorophores. The methodis thus reliable, but time and cost intensive and laborious. Especiallyfor the selection out of very large, diverse cell populations e.g.,apheresis products containing 1×10⁻¹⁰ cells very long sorting times offlow cytometers are unacceptable for an appropriate selection process.Another drawback of FACS™ is that complex and interference-prone flowcytometers can hardly be adapted to a GMP environment necessary forisolating therapeutic cell products. Moreover, the applied pressuresduring the cell selection procedure may compromise cell effectorfunction.

Magnet-assisted isolation of cells is a widely used system for researchand therapeutic application. Although yield and purity of isolated cellsare moderate compared to the FACS™ technology the selection procedure isrobust and does not require sophisticated automatization. The majordrawbacks of the magnet-assisted isolation are the remaining stainingreagents including the magnetic beads on the isolated cells which maycompromise effector function of isolated cell populations. In additionno serial positive selection processes are possible due to theseremaining magnetic reagents on the isolated cells. Serial positiveselection procedures are mandatory for selecting cell populationsdefined by a set of markers. While still making use of a magnetic orfluorescent label, a significant advancement in the isolation of cellsis the “Streptamer® technology that is, for example, described inInternational Patent Application WO 02/054065 and U.S. Pat. No.7,776,562 and in which a receptor binding reagent exhibiting a lowaffinity binding to a receptor located on a surface of cell is used forthe reversible staining and isolation of cells. In contrast to thecurrently used single positive selection combined with magnetic negativeselection (aiming at removal of all cell populations but the one ofinterest) serial positive selection using the Streptamer® technologywith removal of the low affinity receptor binding reagent after eachselection generate cell populations of very high purity and yield.

It is an object of the present invention to provide a method and also anapparatus that overcomes the drawbacks of the known technology forisolation of cells, for example, FACS™ and MACS technology as described.For example, the present invention aims to provide a rapid, efficientand gentle cell selection procedure especially enabling serial positivecell selections for isolating complex cell populations such asregulatory T cells or central memory T-cells for research, diagnosticand especially therapeutic purposes. Ideally, this new method andapparatus should also be suitable for isolation of other complexbiological materials than cells.

This object is solved by the subject matter of the independent claims,inter alia the methods, uses, and arrangements as recited in theindependent claims.

SUMMARY OF THE INVENTION

The present invention provides methods, kits, arrangements a combinationof reagents and the use of a chromatography stationary phase for theisolation of a desired cell, having a known receptor molecule on itssurface, including the separation of such a cell from other cells voidof such receptor on their surface.

According to a first aspect, the invention provides a method ofisolating a target cell, wherein the target cell has a receptor moleculeon the target cell surface, the method comprising:

-   -   providing a sample, the sample comprising the target cell,    -   providing a receptor binding reagent comprising a binding site B        and a binding partner C, wherein the binding site B comprised in        the receptor binding reagent is capable of specifically binding        to the receptor molecule on the target cell surface, wherein the        dissociation constant (K_(D)) for the binding between the        receptor binding reagent via the binding site B and the receptor        molecule is of low affinity or wherein the dissociation rate        constant (koff) for the binding between the receptor binding        reagent via the binding site B and the receptor molecule has a        value of about 3×10⁻⁵ sec⁻¹ or greater, wherein the binding        partner C comprised in the receptor binding reagent is capable        of reversibly binding to a binding site Z of an affinity        reagent, and    -   exposing the sample to chromatography on a suitable stationary        phase, the stationary phase having the affinity reagent        immobilized thereon,    -   wherein the affinity reagent comprises a binding site Z, wherein        said binding site Z forms a reversible bond with the binding        partner C comprised in the receptor binding reagent, and wherein        the binding site B of the receptor binding reagent binds to a        receptor molecule on the target cell surface, thereby reversibly        immobilizing the target cell on the stationary phase.

According to a second aspect the invention provides a method ofisolating a target cell, wherein the target cell has a receptor moleculeon the target cell surface, the method comprising:

-   -   providing a sample, the sample comprising the target cell and a        receptor binding reagent, the receptor binding reagent        comprising a binding site B and a binding partner C, wherein the        binding site B comprised in the receptor binding reagent is        capable of specifically binding to the receptor molecule, and    -   exposing the sample to chromatography on a suitable stationary        phase, the stationary phase being a gel filtration matrix and/or        affinity chromatography matrix, wherein the gel filtration        and/or affinity chromatography matrix comprises an affinity        reagent, wherein the affinity reagent comprises a binding site Z        specifically binding to the binding partner C comprised in the        receptor binding reagent, thereby isolating the target cell.

According to a third aspect the invention provides a method ofchromatographically isolating a target cell from a sample, wherein thetarget cell has a receptor molecule on the target cell surface, themethod comprising:

-   -   providing a sample, the sample comprising the target cell,    -   providing a receptor binding reagent comprising a binding site B        and a binding partner C,    -   wherein the binding site B comprised in the receptor binding        reagent is capable of specifically binding to the receptor        molecule on the target cell surface,    -   wherein the binding partner C comprised in the receptor binding        reagent is capable of reversibly binding to a binding site Z of        an affinity reagent, and    -   exposing the sample to chromatography on a suitable stationary        phase, the stationary phase having the affinity reagent        immobilized thereon,    -   wherein the affinity reagent comprises a binding site Z, wherein        said binding site Z forms a reversible bond with the binding        partner C comprised in the receptor binding reagent, and wherein        the binding site B of the receptor binding reagent binds to a        receptor molecule on the target cell surface, thereby reversibly        immobilising the target cell on the stationary phase,    -   providing a competition reagent, the competition reagent        comprising a binding site, specifically binding to the binding        sites Z of the affinity reagent;    -   loading the competition reagent onto the first stationary phase,        thereby allowing disruption of non-covalent reversible complexes        formed between (a plurality of) the receptor binding reagent,        the receptor molecule and the affinity reagent;    -   recovering an elution sample from the eluate of the first        stationary phase, wherein the elution sample comprises the        target cell;    -   exposing the elution sample to chromatography on a second        suitable stationary phase, the second stationary phase being a        gel filtration matrix and/or affinity chromatography matrix,        wherein the gel filtration and/or affinity chromatography matrix        comprises an affinity reagent having binding sites Z        specifically binding to the binding partner C comprised in the        receptor binding reagent, and    -   passing the elution sample through the second chromatography        column.

According to a fourth aspect the invention provides the use of areceptor binding reagent and/or an affinity reagent for the isolation ofa target cell via chromatography using a stationary phase, wherein thetarget cell has a receptor molecule on the target cell surface, whereinthe receptor binding reagent comprises a binding site B and a bindingpartner C, the binding site of the receptor binding reagent is able tospecifically bind to the receptor molecule of the target cell, whereinthe dissociation constant (KD) for the binding between the receptorbinding reagent via the binding site B and the receptor molecule is oflow affinity or wherein the dissociation rate constant (koff) for thebinding between the receptor binding reagent via the binding site B andthe receptor molecule has a value of about 3×10⁻⁵ sec⁻¹ or greater, andwherein the binding partner C comprised in the receptor binding reagentis able to reversibly bind to a binding site Z of the affinity reagent.

According to a fifth aspect the invention provides the use of one ofstreptavidin, a streptavidin mutein (analog), avidin and an avidinanalogue for isolation of a target cell via chromatography, wherein thechromatography is a gel filtration chromatography.

According to a sixth aspect the invention provides the use of achromatography matrix of one of a cellulose membrane, a plasticmembrane, a polysaccharide gel, a polyacrylamide gel, an agarose gel,polysaccharide grafted silica, polyvinylpyrrolidone grafted silica,polyethylene oxide grafted silica, poly(2-hydroxyethylaspartamide)silica, poly(N-isopropylacrylamide) grafted silica, astyrene-divinylbenzene gel, a copolymer of an acrylate or an acrylamideand a diol, a co-polymer of a polysaccharide andN,N′-methylenebisacrylamide and a combination of any two or more thereoffor the separation of cells, the cells containing a cell nucleus.

According to a seventh aspect the invention provides an arrangement of afirst and a second stationary phase for chromatography,

-   -   wherein the first stationary phase is suitable for cell        separation, the first stationary phase being defined by an        affinity chromatography matrix, wherein the affinity        chromatography matrix has an affinity reagent immobilized        thereon, wherein the affinity reagent has at least one binding        site Z capable of reversibly binding to a binding partner C        comprised in a receptor binding reagent,    -   wherein the second stationary phase is suitable for separation        of target cells from other components, the second stationary        phase being a gel filtration matrix and/or affinity        chromatography matrix, wherein the affinity chromatography        matrix, or the gel filtration and affinity chromatography matrix        comprises an affinity reagent having a binding site Z        specifically binding to said binding partner C comprised in the        receptor binding reagent. In some embodiments, the affinity        reagent comprised in/immobilized on the first stationary phase        and the secondary stationary phase are identical. In some        embodiments, the affinity reagent comprised in/immobilized on        the first stationary phase and the secondary stationary phase        are streptavidin, a streptavidin mutein, avidin or an avidin        mutein.

According to an eight aspect the invention provides a kit for isolatinga target cell, wherein the target cell has a receptor molecule on thetarget cell surface, the kit comprising

-   -   (a) a receptor binding reagent comprising a binding site B and a        binding partner C, wherein the binding site B comprised in the        receptor binding reagent is able to specifically bind to the        receptor molecule of the target cell surface, and wherein the        binding partner C comprised in the receptor binding reagent is        capable of reversibly binding to a binding site Z on a        multimerization reagent; and    -   (b) a stationary phase suitable for cell separation, the        stationary phase being defined by a gel filtration matrix and/or        an affinity chromatography matrix, wherein the affinity        chromatography matrix, or the gel filtration and affinity        chromatography matrix, comprises an affinity reagent having a        binding site Z capable of reversibly binding to the binding        partner C comprised in the receptor binding reagent.

According to a ninth aspect the invention provides a method of isolatinga target cell, wherein the target cell has a receptor molecule on thetarget cell surface, the method comprising:

-   -   providing a sample, the sample comprising the target cell,    -   providing a receptor binding reagent comprising a monovalent        binding site B and a binding partner C, wherein the receptor        binding reagent is selected from the group of an monovalent        antibody fragment, a proteinaceous binding molecule with        immunoglobulin-like functions, an aptamer and an MHC molecule,    -   wherein the monovalent binding site B comprised in the receptor        binding reagent is capable of specifically binding to the        receptor molecule on the target cell surface, wherein the        binding partner C comprised in the receptor binding reagent is        capable of reversibly binding to a binding site Z of an affinity        reagent, and    -   exposing the sample to chromatography on a suitable stationary        phase, the stationary phase having the affinity reagent        immobilized thereon, wherein the affinity reagent comprises a        binding site Z, wherein said binding site Z forms a reversible        bond with the binding partner C comprised in the receptor        binding reagent, and wherein the binding site B of the receptor        binding reagent binds to a receptor molecule on the target cell        surface, thereby reversibly immobilizing the target cell on the        stationary phase.

According to a tenth aspect the invention provides an apparatus forpurification of target cells, the apparatus comprising at least onearrangement of a first and a second stationary phase for chromatography.The first stationary phase of this arrangement is suitable for cellseparation, wherein the first stationary phase is an affinitychromatography matrix, wherein the affinity chromatography matrix has anaffinity reagent immobilized thereon, wherein the affinity reagent hasat least one binding site Z capable of reversibly binding to a bindingpartner C comprised in a receptor binding reagent. The second stationaryphase is suitable for cell separation, wherein the second stationaryphase is a gel filtration matrix and/or affinity chromatography matrix.The affinity chromatography matrix or the gel filtration and affinitychromatography matrix comprises an affinity reagent having a bindingsite Z specifically binding to said binding partner C comprised in thereceptor binding reagent.

According to an eleventh aspect the invention provides A method ofscreening of a target cell for recombinant expression of a desiredreceptor molecule on the target cell surface, wherein the desiredreceptor molecule is to be expressed on the target cell surface, themethod comprising:

-   -   providing a sample, the sample comprising the target cell        suspected of recombinant expression of the desired target        receptor,    -   providing a receptor binding reagent comprising a binding site B        and a binding partner C, wherein the binding site B comprised in        the receptor binding reagent is capable of specifically binding        to the desired receptor molecule on the target cell surface,        wherein the binding partner C comprised in the receptor binding        reagent is capable of reversibly binding to a binding site Z of        an affinity reagent, and    -   exposing the sample to chromatography on a suitable stationary        phase, the stationary phase having the affinity reagent        immobilized thereon,    -   wherein the affinity reagent comprises a binding site Z, wherein        said binding site Z forms a reversible bond with the binding        partner C comprised in the receptor binding reagent, and wherein        the binding site B of the receptor binding reagent binds to a        receptor molecule on the target cell surface, thereby reversibly        immobilizing the target cell on the stationary phase.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the accompanying drawings. The figures illustrateembodiments of methods of the invention. Without wishing to be bound bytheory, the figures include conclusions with regard to the underlyingseparation mechanism. The conclusions are of given for illustrativepurposes only and merely serve in allowing a visualization of how thesurprising separation achievable might be envisaged on a molecularlevel.

FIG. 1 depicts an embodiment of a method of isolating a target cell (2)that has a receptor molecule (4) on the target cell surface (meaning thetarget cell is defined by the presence of at least one common specificreceptor molecule (4)). The sample containing the target cell may alsocontain additional cells (22) that are devoid of the receptor molecule(4) but instead have different receptor molecules (44) on their surface.A receptor binding reagent (1) is provided, for example in the samplethat contains the target cell. The receptor binding reagent (1) has abinding site B (3), which specifically binds to the receptor molecule(4). The receptor binding reagent (1) also includes a binding partner C(5), which can specifically and reversibly bind to a binding site Z (6)of an affinity reagent (8). In some embodiments, the receptor bindingreagent may have a monovalent binding site B and might be a monovalentantibody fragment (for example, a Fab fragment, a single chain Fvfragment or an Fv fragment) or a proteinaceous binding molecule withimmunoglobulin-like functions, an aptamer or an MHC molecule. In thiscontext, it is noted that the affinity reagent used in the presentinvention can also have two or more binding sites Z that can be bound bythe binding partner C, thereby providing a multimerization of thereceptor binding reagent. This affinity reagent used herein can thusalso be a multimerization reagent. The affinity reagent may, forexample, be streptavidin, a streptavidin mutein, avidin, an avidinmutein or a mixture thereof. In addition, different chromatographymatrices coupled to different affinity reagents can be layered into acolumn forming a multicomponent system for separation. The sample, whichincludes the receptor binding reagent (1) and the target cell (2) iscontacted with a chromatography matrix (19), on which the affinityreagent (8) is immobilized. The affinity reagent (8) has a plurality ofbinding sites Z (6), which specifically bind to the binding partner C(5), which is comprised in the receptor binding reagent (1). Thereceptor binding reagent (1) binds via the binding partner C to abinding site Z (6) on the affinity reagent (8), thereby immobilizing thetarget cell (2) via the complex that is formed by the one or morebinding sites Z of the affinity reagent and the binding site Z ofreceptor binding reagent on the chromatography matrix (19). As a resultthe sample is being depleted of the target cell (2), the target cell (2)being thus separated from the other components in the sample includingthe receptor binding reagent (1). In this context, it is noted that thereceptor binding reagent (1) can either be included in the sample thatcontains the target cell to be isolated or the receptor binding reagent(1) can be added to the chromatography matrix (19) for binding to themultimerization reagent (8) immobilised thereon before the sample thatcontains the target cell is added (see also the Experimental Section inthis regard). When a cartridge is filled with such an affinitychromatography matrix (19) and is used for the isolation of a targetcell, by means of an affinity chromatography, such a cartridge is alsoreferred as “Selection Cartridge” herein. In this respect it is notedthat this chromatography method can be carried out as columnchromatography or planar chromatography.

FIG. 2 depicts a further embodiment of a method of isolating a targetcell (2) with receptor molecules (4) on the target cell surface. Themethod illustrated in FIG. 2 can be carried out on its own or incombination with the method as illustrated in FIG. 1 (in the lattercase, the method of FIG. 2 is carried out after the method depicted inFIG. 1). A sample used in the method of FIG. 2 includes the target cell(2), a receptor binding reagent (1) and a competition reagent (7). Thereceptor binding reagent (1) has a binding site B (3) that canspecifically bind to the receptor molecule (4). The receptor bindingreagent (1) also includes a binding partner C (5) that can specificallybind to a binding site Z (6) on an affinity reagent (8) (the affinityreagent (8) can be identical to the affinity/multimerization reagent (8)shown in FIG. 1). The affinity reagent (8) has a plurality of bindingsites Z (6), which are able to specifically bind to the binding partnerC (5) that is included in the receptor binding reagent (1). Also thecompetition reagent (7) has a binding site (9) that is able to bind tothe binding site (6) on the affinity reagent (8). It can also be thecase that the entire competition reagent (7) forms the binding site (9).As an example for the case that the entire competition reagent forms thebinding site (9), the competition reagent (7) may be biotin or a biotinderivate having affinity to streptavidin or streptavidin mutein, whilethe binding partner C (5) of the receptor binding reagent (1) may astreptavidin binding peptide being fused to the receptor binding reagent(1). Both the competition reagent (7) and the receptor binding reagent(1) bind to a binding site (6) of the plurality of binding sites Z (6)that are included in the affinity reagent (8). Thereby, the competitionreagent (7) and the receptor binding reagent (1) are immobilized on thechromatography matrix (19). As a result the sample containing the samplecell is being depleted of the competition reagent (7) and the receptorbinding reagent (1). Since both the competition reagent (7) and thereceptor binding reagent (1) bind to affinity reagent that is comprisedon the chromatography matrix (19), the target cell (2) is not bound tothe chromatography matrix and will, for example, pass through a columnin which the chromatography matrix is used as a stationary phase. When acartridge is filled with such a chromatography matrix (19) and is usedfor the depletion/removal of reactants of a sample containing (apopulation of) target cells, such a cartridge is also referred as“Removal Cartridge” herein. In this respect it is noted that thischromatography method can be carried out as column chromatography orplanar chromatography.

FIG. 3 shows an embodiment of a method of separating/isolating a targetcell containing a nucleus (2). A sample is provided which includes thetarget cell (2), and optionally for example, a receptor binding reagent(1) and a competition reagent (7). The sample is loaded onto achromatography column, which includes a gel filtration matrix (19)selected from a matrix using a chromatography matrix selected from thegroup consisting of a polysaccharide gel, a polyacrylamide gel, anagarose gel, polysaccharide grafted silica, polyvinylpyrrolidone graftedsilica, polyethylene oxide grafted silica,poly(-hydroxyethylaspartamide) silica, poly(N-isopropylacrylamide)grafted silica, a styrene-divinylbenzene gel, a copolymer of an acrylateor an acrylamide and a diol, a co-polymer of a polysaccharide andN,N′-methylenebisacrylamide and a combination of any two or morethereof. As the sample is allowed to pass through the gel filtrationmatrix (19), the receptor binding reagent (1) and the competitionreagent (7) remain on the column longer. These reagents may, forexample, enter pores of the gel filtration matrix and the target cell(2) elutes from the chromatography column earlier and can be collectedfor further use.

FIG. 4 depicts a further embodiment of a method of isolating a targetcell (2) that is defined by the presence of at least one common specificreceptor molecule (4) on the target cell surface. In this method a firstchromatography column (a selection cartridge) and a secondchromatography column (a removal cartridge) are employed. A sample isprovided that includes inter alia the target cell (2) with receptormolecules (4) and a further cell (22) with different receptor molecules(44) on its surface. The sample also includes a receptor binding reagent(1), which has a binding site B (3) that specifically binds to thereceptor molecule (4). The receptor binding reagent (1) also includes abinding partner C (5) that specifically binds to a binding site Z (6) onan affinity reagent (8). The sample is loaded onto the firstchromatography column, which has a suitable stationary phase in the formof an affinity chromatography matrix (29), wherein the affinitychromatography matrix (29) has the affinity reagent (8) immobilizedthereon. A non-covalent reversible complex between a plurality of thereceptor binding reagent (1), the affinity (multimerization) reagent (8)and the target cell (2), but not the further cell (22), is formed. Thefurther cell will pass through the first chromatography columnspontaneously or after washing of the chromatography column (theoptional washing step is not shown in FIG. 4). A competition reagent (7)is then loaded onto the chromatography column. The competition reagent(7) has a binding site (9) (or constitutes a binding site) that is ableto bind to the binding site Z (6) of the affinity reagent (8). Aplurality of the competition reagent (7) is present and a portionthereof forms a complex with the affinity reagent (8), and is therebyimmobilized on the chromatography matrix (29). As a result of thiscompetitive binding, the binding of the binding partner C (5), which isincluded in the receptor binding reagent (1), to the binding site Z isdisrupted. By so doing, the receptor binding reagent is released fromthe chromatography matrix (29) and thus also the non-covalent reversiblecomplex formed between the receptor binding reagent (1), the affinityreagent (8) and the target cell (2) disintegrates. An elution samplefrom the eluate of the first chromatography column, which includes thetarget cell (2), the competition reagent (7) and the receptor bindingreagent (1), is collected. The elution sample is loaded onto the secondchromatography column, which has a suitable stationary phase that isboth an affinity chromatography matrix (19) and, at the same time, canact as gel permeation matrix. The affinity chromatography matrix (19)has an affinity reagent (8) immobilized thereon. The affinity reagent(8) may, for example, be streptavidin, a streptavidin mutein, avidin, anavidin mutein or a mixture thereof. The receptor binding reagent (1) andthe competition reagent (7) bind to a binding site Z (6) on the affinityreagent (8), thereby being immobilized on the chromatography matrix(19). As a result the elution sample containing the isolated targetcells is being depleted of the receptor binding reagent (1) and thecompetition reagent (7). The target cells, being freed or any reactants,are now in a condition for further use, for example, for diagnosticapplications (for example, further FACS™ sorting) or for any cell basedtherapeutic application.

FIG. 5 shows the results of an experiment for enriching CD8+ cells fromperipheral blood mononuclear cells (PBMC). This experiment was performedon two columns both containing Sephadex-50 resin coupled withStrep-Tactin® as the affinity reagent and using a CD8 binding Fabfragment as monovalent receptor binding reagent carrying a streptavidinbinding peptide as binding partner C. Diagrams B-D show the results ofan isolation according to a method of the invention while diagrams E-Gshow the result for a negative control.

FIG. 6A and FIG. 6B are both schematic drawings of an embodiment of anapparatus of the invention for the isolation of cells using at least onesequential arrangement of a selection cartridge and a removal cartridge.The apparatus 10 of FIG. 6A contains a peristaltic pump 102 and variousvalves (for example, magnetic valves) that control the flow of theliquid phases (sample buffer, washing buffer, eluent) that are used inthe chromatographic isolation of target cells. The peristaltic pump andthe valves are controlled by a microprocessor (not shown). Theindividual reservoirs and cartridges of the apparatus 10 are fluidlyconnected to each via tubings 400. The apparatus 10 contains a bufferreservoir 114 that is fluidly connected via a sample inlet such a tube400 to a sample reservoir 116 that contains a sample (for example bloodor other body cells) including target cells that are to be purified. Thecell sample contained in a suitable buffer is then applied to the firstselection cartridge 104 that contains a suitable stationary phase asexplained in FIG. 3 in the form of an affinity chromatography matrixwith an affinity reagent immobilized thereon. In the selection cartridgetarget cells carrying a first kind of specific common receptor moleculeare immobilized by means of a receptor binding reagent specificallybinding the first kind of receptor molecules. Cells that do not carrythe first kind of receptor molecule flow through the column and arediscarded via a waste reservoir 112. An eluent (a competition agent asexplained herein) stored in an elution buffer reservoir 110 is thenapplied on the column, leading to the disruption of the reversible bondformed between the affinity reagent and the receptor binding reagent andthus also to the elution of the target cells. The eluate containing thetarget cells is then applied to a removal cartridge 106 that contains,as explained in FIG. 3, a second stationary phase on which an affinityreagent is present. While the affinity reagent captures/immobilizes thereceptor binding reagent and the competition reagent, the purifiedtarget cells pass through this column and are directed to a secondarrangement of a selection cartridge 204 and a removal cartridge 206.The target cells are purified in this second arrangement via a secondkind of common specific receptor molecule as explained above, with cellsthat do not carry the second kind of receptor molecule on their surfaceflowing through the selection cartridge and being discarded via a secondwaste reservoir 212. In FIG. 6A the elution buffer reservoir 110 that isfluidly connected to the selection cartridge 204 of the secondsequential arrangement of selection cartridge and removal cartridge, isdepicted as an additional reservoir to the one connected to theselection cartridge 104. However, in case the same competition reagentis used, the apparatus 10 can comprise only a single elution bufferreservoir that is fluidly connected to the selection cartridge of eachof the plurality of “cartridge arrangements”. Finally, the removalcartridge 206 is fluidly connected to a sample outlet 214 for collectionof the isolated target cells. The apparatus of FIG. 6B has a similardesign with three serially connected “cartridge arrangements” eachconsisting of a selection cartridge and a removal cartridge. Theapparatus of FIG. 6B also includes a temperature control element formaintaining a constant temperature such as 4° C., 15° C. or 25° C.

FIGS. 7A to 7C show the results of a further experiment for enrichinghuman CD8+ cells from peripheral blood mononuclear cells (PBMC) CD8+cells, with FIG. 7A showing the starting sample of the PBMC's, FIG. 7Bshowing the CD8+ cell negative wash fraction and FIG. 7C showing theCD8+ positive eluate fraction.

FIGS. 8A to 8C show the results of an experiment for enriching humanCD8+ cells from whole blood with FIG. 8A showing the starting wholeblood sample, FIG. 8B showing the CD8+ cell negative wash fraction andFIG. 8C showing the CD8+ positive eluate fraction.

FIGS. 9A to 9C show the results of an experiment for enriching murineCD4+ cells from splenocytes with FIG. 9A showing the starting sample ofthe splenocytes, FIG. 9B showing the CD4+ cell negative wash fractionand FIG. 9C showing the CD4+ positive eluate fraction.

FIGS. 10A to 10C show the results of an experiment for enriching humanCD4+ cells from peripheral blood mononuclear cells (PBMC) with FIG. 10Ashowing the starting sample of the PBMC's, FIG. 10B showing the CD4+cell negative wash fraction and FIG. 10C showing the CD4+ positiveeluate fraction.

FIGS. 11A to 11C show the results of an experiment for enriching humanCD4+ cells from whole blood, with FIG. 11A showing the starting wholeblood sample, FIG. 11B showing the CD4+ cell negative wash fraction andFIG. 11C showing the CD4+ positive eluate fraction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and an apparatus of performing afluid chromatographic separation of cells and other biologic entitiessuch as cell organelles, viruses, liposomes and the like (the referenceto target cells in the following thus also includes a reference to allother biological entities). A target cell or a population of targetcells is isolated from a sample that, for example, may include a varietyof different cells or cell populations. Virtually any said target cellthat has at least one common receptor molecule on its surface can beseparated from other components contained in a sample. In order toachieve an avidity effect, as discussed below, for affinitychromatography as described herein, the receptor molecule is typicallypresent in two or more copies on the surface of the target cell. Theterm “(target) cell” as used herein encompasses all biologicalentities/vesicles in which a membrane (which can also be a lipidbilayer) separates the interior from the outside environment (ambience)and which comprise one or more kinds of specific receptor molecule(s) onthe surface of the biological entity/vesicle. This means the targetcell/biological entity/vesicle or the population of target cells isdefined by the presence of at least one common specific receptormolecule on the surface. “Isolation” as used herein means that thetarget cell is enriched in a sample that is obtained as a result of amethod of the invention compared to the content (concentration) of thesample that was for the isolation of the target cell. This means thetarget cell might be enriched in a sample, for example from about acontent of about 0.1% of the entire amount of cells in a sample to sayabout 10% or more, or 20% or more, 30% or more, 40% or more, in a samplecollected from a method of the invention. “Isolated” also means that thesample obtained contains the target cell as essentially only kind ofcell (cell population), for example, the target cells represents morethan 75%, or more than 80%, or more than 85%, or more than 90%, or morethan 95% or more than 97% or more than 99% of the cells present in asample. “Isolated” also includes that a sample containing the targetcell is devoid of reactants (for example, receptor binding reagents orcompetition reagents as defined herein) after having undergone anisolation/purification method of the invention. The term “isolation”also includes the detection of the presence of non-presence of targetcells in a sample. Accordingly, the isolation of target cells of can beused either for analytical or preparative purposes (for example, fordetecting the presence of a target cell population but also forquantification of cells present in a sample or for isolation of cells ona large scale for cell-based therapy). Analytical purposes includediagnostic applications as well as applications in basic research inwhich for example, an isolation method of the invention is used forscreening purposes, for example, whether a particular receptor molecule,for example, a G-protein coupled receptor (GPCR) or any otherphysiologically relevant receptor (e.g. insulin receptor) isrecombinantly expressed in a chosen host cells (see also below).

In some embodiments the cell may be a prokaryotic cell, such as abacterial cell. The cell may in some embodiments be an archaeon. Thecell may in some embodiments be a virus or an organelle such as amitochondrion, a chloroplast, a microsome, a lysosome, a Golgi apparatusor a nucleus. In some embodiments the cell may be an eukaryotic cell,such as a plant cell, a fungal cell, a yeast cell, a protozoon or ananimal cell. The target cell includes in some embodiments a cellnucleus. In some embodiments the target cell is a mammalian cell,including a cell of a rodent species, or an amphibian cell, e.g. of thesubclass Lissamphibia that includes e.g. frogs, toads, salamanders ornewts. Examples of a mammalian cell include, but are not limited to, ablood cell, a semen cell or a tissue cell, e.g. a hepatocyte or a stemcell, e.g. CD34-positive peripheral stem cells or Nanog or Oct-4expressing stem cells derived from a suitable source. A blood cell mayfor instance be a leukocyte or an erythrocyte. A leukocyte may forexample be a neutrophil, an eosinophil, a basophil, a monocyte, alymphocyte, a macrophage or a dendritic cell. A respective lymphocytemay for example be a T cell—including a CMV-specific CD8+T-lymphocyte, acytotoxic T-cell a, memory T-cell (an illustrative example of memoryT-cells are CD62L⁺CD8⁺ specific central memory T-cells) or a regulatoryT-cell (an illustrative example of Treg are CD4⁺CD25⁺CD45RA+ Tregcells), a T-helper cell, for example, a CD4⁺ T-helper cell, a B cell ora natural killer cell, to mention only a few illustrative examples.

The fact that the target cell population or, as mentioned above, anyother population of a biological entity in which a membrane (which canalso be a lipid bilayer) separates the interior from the outsideenvironment and that is further characterized to comprise a commonspecific receptor molecule on the surface can be purified by the methodsof the invention under subsequent removal of any used purificationreagent (receptor binding reagent; competition reagent,affinity/multimerization reagent) offers—beyond the advantage that, ifthe target is a cell or an organelle, the physiological status is notaltered—the regulatory advantage that the purification reagents are notadministered to the patient during the use of such purified biologicalentities as medicaments. In such cases, regulatory authorities like FDA(USA) or EMEA (Europe) require less expensive constraints with respectto production processes for said purification reagents than in caseswhere the purification reagent is administered together with themedicament being a cell or a liposome. Therefore, a clear technicaladvantage exists also with respect to the methods of the invention forthe purification of entities of which no physiological status can bemanipulated like for liposomes, for example, if such liposomes have tobe purified and are used as medicaments.

Examples of mammals include, but are not limited to, a rat, a mouse, arabbit, a guinea pig, a squirrel, a hamster, a hedgehog, a cat, aplatypus, an American pika, an armadillo, a dog, a lemur, a goat, a pig,an opossum, a horse, an elephant, a bat, a woodchuck, an orang-utan, arhesus monkey, a woolly monkey, a macaque, a chimpanzee, a tamarin(saguinus oedipus), a marmoset and a human. The cell may for instance bea cell of a tissue, such as an organ or a portion thereof. Examples of arespective organ include, without being limited thereto, adrenal tissue,bone, blood, bladder, brain, cartilage, colon, eye, heart, kidney,liver, lung, muscle, nerve, ovary, pancreas, prostate, skin, smallintestine, spleen, stomach, testicular, thymus, tumour, vascular oruterus tissue, or connective tissue. In some embodiments the cell is astem cell.

A sample from which the target cell is to be isolated may be of anyorigin. It may for instance, but not limited to, be derived from humans,animals, plants, bacteria, fungi, or protozoae. Accordingly, any of thefollowing samples selected from, but not limited to, the groupconsisting of a soil sample, an air sample, an environmental sample, acell culture sample, a bone marrow sample, a rainfall sample, a falloutsample, a sewage sample, a ground water sample, an abrasion sample, anarchaeological sample, a food sample, a blood sample (including wholeblood), a serum sample, a plasma sample, an urine sample, a stoolsample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluidsample, a nasopharyngeal wash sample, a sputum sample, a mouth swabsample, a throat swab sample, a nasal swab sample, a bronchoalveolarlavage sample, a bronchial secretion sample, a milk sample, an amnioticfluid sample, a biopsy sample, a cancer sample, a tumour sample, atissue sample, a cell sample, a cell culture sample, a cell lysatesample, a virus culture sample, a nail sample, a hair sample, a skinsample, a forensic sample, an infection sample, a nosocomial infectionsample, a space sample or any combination thereof. Where desired, arespective sample may have been preprocessed to any degree. As anillustrative example, a tissue sample may have been digested,homogenised or centrifuged prior to being used in a method according tothe present invention. In another illustrative example, a sample of abody fluid such as blood might be obtained by standard isolation ofblood cells. If an isolation method described here is used in basicresearch, the sample might be cells of in vitro cell cultureexperiments. The sample will typically have been prepared in form of afluid, such as a solution or dispersion.

Generally, a chromatographic method according to the invention is afluid chromatography, typically a liquid chromatography. Thechromatography can be carried out in a flow through mode in which afluid sample containing the cells to be isolated is applied, forexample, by gravity flow or by a pump on one end of a column containingthe chromatography matrix and in which the fluid sample exists thecolumn at the other end of the column (cf. also Examples 1 to 7 in thisregard). In addition the chromatography can be carried out in an “up anddown” mode in which a fluid sample containing the cells to be isolatedis applied, for example, by a pipette on one end of a column containingthe chromatography matrix packed within a pipette tip and in which thefluid sample enters and exists the chromatography matrix/pipette tip atthe other end of the column (cf. Examples 8 to 10 in this regard).Alternatively, the chromatography can also be carried out in a batchmode in which the chromatography material (stationary phase) isincubated with the sample that contains the cells, for example, undershaking, rotating or repeated contacting and removal of the fluidsample, for example, by means of a pipette. Any material may be employedas chromatography matrix in the context of the invention, as long as thematerial is suitable for the chromatographic isolation of cells. Asuitable chromatography material is at least essentially innocuous, i.e.not detrimental to cell viability (or the viability or stability of thebiological entity), when used in a packed chromatography column underdesired conditions for cell isolation and/or cell separation. Achromatography matrix as used in the present invention remains in apredefined location, typically in a predefined position, whereas thelocation of the sample to be separated and of components includedtherein, is being altered. Thus, the chromatography matrix is a“stationary phase” in line with the regular understanding of the personskilled in the art that the stationary phase is the part of achromatographic system through which the mobile phase flows (either byflow through or in a batch mode) and where distribution of thecomponents contained in the liquid phase (either dissolved or dispersed)between the phases occurs. The terms “chromatography matrix” and“stationary phase” are thus used interchangeable herein. In this regard,it is noted that particles such as freely movable magnetic beads thatare added to a liquid sample, mixed with the sample and are then removedfrom the sample, for example, by discarding the supernatant (liquid)while holding the beads temporarily in place (for example, by anexternal magnetic or by centrifugation) are not a stationary phase asused herein. Thus, a method in which such (magnetic) beads are added toa sample containing the target cells for immobilization of the targetcells (via a complex formed between the target cells, the receptorbinding reagent and the affinity/multimerization reagent) on such beads,and the beads are then separated from the sample, for example bytemporarily holding the beads in place, while discarding thesupernatant, is not a method of the invention.

Typically, the respective chromatography matrix has the form of a solidor semi-solid phase, whereas the sample that contains the target cell tobe isolated/separated is a fluid phase. The mobile phase used to achievechromatographic separation is likewise a fluid phase. The chromatographymatrix can be a particulate material (of any suitable size and shape) ora monolithic chromatography material, including a paper substrate ormembrane (cf. the Example Section). Thus, the chromatography can be bothcolumn chromatography as well as planar chromatography. In addition tostandard chromatography columns, columns allowing a bidirectional flowsuch as PhyTip® columns available from PhyNexus, Inc. San Jose, Calif.,U.S.A. or pipette tips can be used for column based/flow through modebased chromatographic separation of cells as described here. Thus,pipette tips or columns allowing a bidirectional flow are alsoencompassed by the term “chromatography columns” as used herein. If aparticulate matrix material is used, the particulate matrix materialmay, for example, have a mean particle size of about 5 μm to about 200μm, or from about 5 μm to about 400 μm, or from about 5 μm to about 600μm. As explained in detail the following, the chromatography matrix may,for example, be or include a polymeric resin or a metal oxide or ametalloid oxide. If planar chromatography is used, the matrix materialmay be any material suitable for planar chromatography, such asconventional cellulose-based or organic polymer based membranes (forexample, a paper membrane, a nitrocellulose membrane or a polyvinylidenedifluoride (PVDF) membrane) or silica coated glass plates. In oneembodiment, the chromatography matrix/stationary phase is a non-magneticmaterial or non-magnetisable material.

Non-magnetic or non-magnetisable chromatography stationary phases thatare used in the art, and that are also suitable in the presentinvention, include derivatized silica or a crosslinked gel. Acrosslinked gel (which is typically manufactured in a bead form) may bebased on a natural polymer, i.e. on a polymer class that occurs innature. For example, a natural polymer on which a chromatographystationary phase is based is a polysaccharide. A respectivepolysaccharide is generally crosslinked. An example of a polysaccharidematrix is an agarose gel (for example, Superflow™ agarose or aSepharose® material such as Superflow™ Sepharose® that are commerciallyavailable in different bead and pore sizes) or a gel of crosslinkeddextran(s). A further illustrative example is a particulate cross-linkedagarose matrix, to which dextran is covalently bonded, that iscommercially available (in various bead sizes and with various poresizes) as Sephadex® or Superdex®, both available from GE Healthcare.Another illustrative example of such a chromatography material isSephacryl® which is also available in different bead and pore sizes fromGE Healthcare.

A crosslinked gel may also be based on a synthetic polymer, i.e. on apolymer class that does not occur in nature. Usually such a syntheticpolymer on which a chromatography stationary phase for cell separationis based is a polymer that has polar monomer units, and which istherefore in itself polar. Such a polar polymer is hydrophilic.Hydrophilic (“water-loving”) molecules, also termed lipophobic (“fathating”), contain moieties that can form dipole-dipole interactions withwater molecules. Hydrophobic (“water hating”) molecules, also termedlipophilic, have a tendency to separate from water.

Illustrative examples of suitable synthetic polymers arepolyacrylamide(s), a styrene-divinylbenzene gel and a copolymer of anacrylate and a diol or of an acrylamide and a diol. An illustrativeexample is a polymethacrylate gel, commercially available as aFractogel®. A further example is a copolymer of ethylene glycol andmethacrylate, commercially available as a Toyopearl®. In someembodiments a chromatography stationary phase may also include naturaland synthetic polymer components, such as a composite matrix or acomposite or a co-polymer of a polysaccharide and agarose, e.g. apolyacrylamide/agarose composite, or of a polysaccharide andN,N′-methylenebisacrylamide. An illustrative example of a copolymer of adextran and N,N′-methylenebisacrylamide is the above-mentionedSephacryl® series of material. A derivatized silica may include silicaparticles that are coupled to a synthetic or to a natural polymer.Examples of such embodiments include, but are not limited to,polysaccharide grafted silica, polyvinyl-pyrrolidone grafted silica,polyethylene oxide grafted silica, poly(-hydroxyethylaspartamide) silicaand poly(N-isopropylacrylamide) grafted silica.

A chromatography matrix employed in the present invention is in someembodiments a gel filtration (also known as size exclusion) matrix, forexample, when used in a removal cartridge as described herein. A gelfiltration can be characterized by the property that it is designed toundergo, at least essentially, no interaction with the cells to beseparated. Hence, a gel filtration matrix allows the separation of cellsor other biological entities as defined herein largely on the basis oftheir size. A respective chromatography matrix is typically aparticulate porous material as mentioned above. The chromatographymatrix may have a certain exclusion limit, which is typically defined interms of a molecular weight above which molecules are entirely excludedfrom entering the pores. The respective molecular weight defining thesize exclusion limit may be selected to be below the weightcorresponding to the weight of a target cell (or biological entity) tobe isolated. In such an embodiment the target cell is prevented fromentering the pores of the size exclusion chromatography matrix.Likewise, a stationary phase that is an affinity chromatography matrixmay have pores that are of a size that is smaller than the size of achosen target cell. In illustrative embodiments the affinitychromatography matrix and/or the gel filtration matrix has a mean poresize of 0 to about 500 nm.

Other components present in a sample such as receptor binding moleculesor a competition reagent may have a size that is below the exclusionlimit of the pores and this can enter the pores of the size exclusionchromatography matrix. Of such components that are able to partially orfully enter the pore volume, larger molecules, with less access to thepore volume will usually elute first, whereas the smallest moleculeselute last. In some embodiments the exclusion limit of the sizeexclusion chromatography matrix is selected to be below the maximalwidth of the target cell. Hence, components that have access to the porevolume will usually remain longer in/on the size exclusionchromatography matrix than target cell. Thus, target cells can becollected in the eluate of a chromatography column separately from othermatter/components of a sample. Therefore components such as a receptorbinding reagent, or where, applicable a competition reagent, elute at alater point of time from a gel filtration matrix than the target cell.This separation effect will be further increased, if the gel permeationmatrix comprises an affinity reagent (usually covalently bound thereon)that comprises binding sites, for example binding sites Z that are ableto bind reagents such as a receptor binding reagent and/or a competitionreagent present in a sample. The receptor binding reagent and/or thecompetition reagent will be bound by the binding sites Z of the affinityreagent and thereby immobilized on the gel permeation matrix. Thismethod is usually carried out in a removal cartridge as used in thepresent invention and in some embodiments a method, a combination and akit according to the invention include and/or employ such a gelfiltration matrix. In a respective method cells are accordinglyseparated on the basis of size.

A chromatography matrix employed in the present invention may alsoinclude magnetically attractable matter such as one or more magneticallyattractable particles or a ferrofluid. A respective magneticallyattractable particle may comprise a multimerization reagent or anaffinity reagent with binding site that is capable of binding a targetcell. Magnetically attractable particles may contain diamagnetic,ferromagnetic, paramagnetic or superparamagnetic material.Superparamagnetic material responds to a magnetic field with an inducedmagnetic field without a resulting permanent magnetization. Magneticparticles based on iron oxide are for example commercially available asDynabeads® from Dynal Biotech, as magnetic MicroBeads from MiltenyiBiotec, as magnetic porous glass beads from CPG Inc., as well as fromvarious other sources, such as Roche Applied Science, BIOCLON, BioSourceInternational Inc., micromod, AMBION, Merck, Bangs Laboratories,Polysciences, or Novagen Inc., to name only a few. Magneticnanoparticles based on superparamagnetic Co and FeCo, as well asferromagnetic Co nanocrystals have been described, for example byBitten, A. et al. (J. Biotech. (2004), 112, 47-63). However, in someembodiments a chromatography matrix employed in the present invention isvoid of any magnetically attractable matter.

In some embodiments of a method of isolating a target cell, achromatography matrix is employed as an affinity chromatography matrix.An affinity chromatography matrix itself includes permanently bonded(usually covalently bonded) moieties that are capable to specificallybind a selected target. For example, a conventional affinitychromatography matrix may include an antibody that binds a particulargiven target. Alternatively, a chromatography matrix that is used forImmobilized Metal-chelate Affinity Chromatography (IMAC) is modifiedwith a chelating ligand agent such as tridentate iminodiacetic acid tobe able to form coordination bonds between metal ions and certainexposed side chains of a protein or with oligohistidine tags, forexample. Thus, in the art an affinity chromatography matrix is generallydesigned such that itself is able to specifically bind the analyte ortarget that is to be isolated. In the present invention, in which thechromatography matrix is used, either, for example, in a “selectioncartridge” as explained in more detail below, the affinitychromatography matrix itself is not designed to be capable ofspecifically binding the target cell that is to be isolated. Rather, insuch embodiments the affinity chromatography matrix (stationary phase)used in the present invention comprises an affinity reagent that has atleast one or more binding sites Z that are able to specifically bind toa receptor binding reagent that is also employed in the presentinvention. When the receptor binding reagent is brought into contactwith the affinity/multimerization reagent, a reversible complex via thebinding partner C of the receptor binding reagent and the one or morebinding sites Z of the affinity/multimerization reagent is formed. Thus,this complex formation relies on non-covalent interactions between aligand and its respective binding partner and is thus fundamentallydifferent from the use of cleavable covalent bonds as described inBonnafous et al, supra. It is usually sufficient that the affinityreagent contains one binding site Z that is able to form a reversiblebond with the binding partner C, as long as the affinity reagent ispresent/provided on the affinity chromatography matrix in a sufficientlyhigh surface density to cause an avidity effect when the complex betweenthe receptor binding reagent and the affinity reagent is formed via thebinding site and the binding partner C. However, it is also possiblethat the affinity reagent comprises two or more binding sites Z for thebinding partner C. In the then non-covalent binding complex formed, twoor more receptor binding reagents are immobilized on the affinitychromatography matrix closely arranged to each other such that anavidity effect can take place if a target cell having (at least twocopies of) a receptor molecule is present in the sample, is brought intocontact with the receptor binding reagent that have one or more bindingsites B being able to bind the particular receptor molecule. Thus, inthese embodiments an avidity (multimerization) effect similar to the onedescribed in U.S. Pat. Nos. 7,776,562, 8,298,782 or International Patentapplication WO02/054065 can take place for allowing a reversibleimmobilization of the target cells on the affinity chromatographymatrix. Since the bond between the binding sites Z of the affinityreagent (that then may also act as multimerization agent) and thebinding partner C of the receptor binding reagent can be disrupted byaddition of a competition agent, the target cells can be subsequentlyeluted under mild conditions under which the receptor binding reagentcompletely dissociates from the target cell, thereby avoiding that thereceptor binding reagent affects the functional status of the targetcell. This isolation of target cells via this affinity chromatographymethod thus does not only have the advantage that it allows for theisolation/purification of target cell population (or any otherbiological entity described herein) without altering the functionalstatus of the target cell population that is defined by a commonspecific receptor molecule. Rather, this method also has the addedadvantage that it entirely abolishes the need to use magnetic beads forcell purification and thereby simplifies any further handling of thecell and opens the way to automatization of the isolation of targetcells, as also described herein.

In other embodiments of a method according to the invention achromatography matrix is used that has an affinity reagent immobilizedthereon. The affinity reagent is able to bind a binding partner C thatis included in a receptor binding reagent (see below). Such achromatography matrix may be an affinity chromatography matrix. It mayalso be a gel filtration matrix, to which the affinity reagent has beencoupled. The chromatography matrix is in some embodiments included in achromatography column, for example packed therein. By means of theimmobilized affinity reagent the chromatography matrix can deplete amobile phase of the receptor binding reagent. A sample that is contactedwith the chromatography matrix, for example, loaded onto a column packedtherewith, can likewise be depleted of the receptor binding reagent. Inone method according to the invention the receptor binding reagent isincluded in a sample that is contacted with a respective stationaryphase, i.e. chromatography matrix.

After applying the sample containing the target cell, the chromatographymatrix (regardless of being used for affinity chromatography or for gelpermeation) may subsequently be washed with a mobile phase, such as anaqueous medium, e.g. a buffer, in order to remove any matter that hasnot been immobilized on the chromatography matrix. Dissociation of theabove described non-covalent complex, the formation of which immobilizesthe target cell on the affinity chromatography matrix, may then beinduced, for example, by a change in conditions. Such a change inconditions may for instance be a change in the ionic strength of anaqueous mobile phase or a change in temperature. In some embodiments acompetition reagent is employed in order to induce dissociation of thereversible non-covalent complex between receptor, receptor bindingreagent and affinity reagent. The competition reagent is able toassociate to the affinity reagent by occupying or blocking the bindingsite of the affinity reagent for the binding partner included in thereceptor binding reagent. By using a competition reagent with aparticularly high affinity for the affinity reagent or by using anexcess of the competition reagent relative to at least one of the targetcell and the receptor binding reagent (in this case, the competitionreagent might also have a lower affinity to the binding site Z of theaffinity reagent than the binding partner C of the receptor bindingreagent) the non-covalent bonding between the receptor binding reagentand the multimerization reagent may be disrupted. The target cell isallowed to elute from the chromatography matrix, e.g. from the columninto which the chromatography matrix is packed. The eluate is collectedand the target cell thereby collected.

In some embodiments a source sample is used, which includes or issuspected to include the target cell, and to which the receptor bindingreagent is added in order to allow the formation of the above describednon-covalent complex that involves the target cell and the affinityreagent on the affinity chromatography matrix. As an illustrativeexample, a blood sample (for example a whole blood sample) or a lymphsample may define such a source sample (cf. the Example Section). Areceptor binding reagent may be selected that has a binding site for adesired target cell, which is present in blood or lymph, respectively.The receptor binding reagent, optionally also some buffer, may be addedto the blood sample or the lymph sample. The buffer used may be at leastessentially identical to a buffer used for equilibrating thechromatography matrix and used for subsequent washing. Subsequently, thesample may be loaded onto the chromatography column. This chromatographycolumn may have an affinity reagent immobilized on its matrix, which canbind the receptor binding reagent. Alternatively, the receptor bindingreagent can already be immobilized on the affinity chromatography matrixbefore the sample of the target cell is applied to the affinitychromatography matrix. After the sample, for example, a blood or lymphsample, optionally with the receptor binding reagent has been entirelyloaded onto the chromatography column, the chromatography matrix may bewashed with a mobile phase. A competition reagent, which may be includedin a buffer used for washing of the chromatography matrix, may then beloaded onto the chromatography column. Subsequently the chromatographymatrix may be washed with a mobile phase. The elution of the target cellmay be monitored using standard detection techniques such as an opticaldetection device. The target cell may then be collected. Such an eluatemay thus include a receptor binding reagent and/or a competitionreagent.

In order to be able further purify such an eluate of target cells achromatography matrix (for example a size exclusion chromatographymatrix) may include an affinity reagent, for example, a moleculeimmobilized on the chromatography matrix, that has binding sites Z thatare able to specifically bind to the binding partner B that is includedin the receptor binding reagent and/or to the competition reagent.

Thus, in line with the above, a size exclusion chromatography matrixused herein may have an affinity reagent immobilized thereon. Since arespective chromatography matrix is also able to separate matteraccording to size and/or shape, it can be addressed as a mixed modechromatography matrix. Thus, in embodiments where the affinity reagentimmobilized on such a size exclusion chromatography matrix does notmatch a receptor binding reagent in that the affinity reagent has abinding site, which cannot form a complex with the selected receptorbinding reagent, the mixed mode chromatography matrix can still beemployed as a size exclusion chromatography matrix. In embodiments wherethe immobilized affinity reagent has a binding site, which does have thecapability to form a complex with the selected receptor binding reagent,the affinity reagent can serve in reversibly immobilizing the targetcell on the chromatography matrix.

The fluid phase used as the mobile phase in chromatography may be anyfluid suitable for preserving the biological activity of the targetcell. Typically, the fluid is a liquid. In some embodiments therespective liquid is or includes water, for example in the form of anaqueous solution. Further components may be included in a respectiveaqueous solution, for example dissolved or suspended therein. As anillustrative example an aqueous solution may include one or more buffercompounds. Numerous buffer compounds are used in the art and may be usedto carry out the various processes described herein. Examples of buffersinclude, but are not limited to, solutions of salts of phosphate such asphosphate buffered saline (PBS), carbonate, succinate, carbonate,citrate, acetate, formate, barbiturate, oxalate, lactate, phthalate,maleate, cacodylate, borate, N-(2-acetamido)-2-amino-ethanesulfonate(also called (ACES), N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonicacid (also called HEPES),4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid (also calledHEPPS), piperazine-1,4-bis(2-ethanesulfonic acid) (also called PIPES),(2-[Tris(hydroxymethyl)-methylamino]-1-ethansulfonic acid (also calledTES), 2-cyclohexylamino-ethanesulfonic acid (also called CHES) andN-(2-acetamido)-iminodiacetate (also called ADA). Any counter ion may beused in these salts; ammonium, sodium, and potassium may serve asillustrative examples. Further examples of buffers include, but are notlimited to, tri-ethanolamine, diethanolamine, zwitter-ionic buffers suchas betaine, ethylamine, triethylamine, glycine, glycylglycine,histidine, tris-(hydroxymethyl)aminomethane (also called TRIS),bis-(2-hydroxyethyl)-imino-tris(hydroxymethyl)-methane (also calledBIS-TRIS), and N-[Tris(hydroxymethyl)-methyl]-glycine (also calledTRICINE), to name only a few. The buffer may further include componentsthat stabilize the target cell to be isolated, for example proteins suchas (serum) albumin, growth factors, trace elements and the like. Thechoice of the suitable mobile phase is within the knowledge of theperson of average skill in the art and can be carried out empirically.

In line with the co-pending International Patent ApplicationPCT/EP2012/063969, published as WO 2013/011011, (the entire content ofwhich is incorporated herein by reference for all purpose) the strengthof the binding between the receptor binding reagent and a receptormolecule on a target cell may not be not essential for the reversibilityof the binding of the target cell to the affinity reagent via thereceptor binding reagent. Rather, irrespective of the strength of thebinding, meaning whether the dissociation constant (K_(d)) for thebinding between the receptor binding reagent via the binding site B andthe receptor molecule is of low affinity, for example, in the range of aK_(d) of about 10⁻³ to about 10⁻⁷ M, or of high affinity, for example,in the range of a K_(d) of about 10⁻⁷ to about 1×10⁻¹⁰ M, a target cellcan be reversibly stained as long as the dissociation of the binding ofthe receptor binding reagent via the binding site B and the receptormolecule occurs sufficiently fast. In this regard the dissociation rateconstant (k_(off)) for the binding between the receptor binding reagentvia the binding site B and the receptor molecule may have a value ofabout 3×10⁻⁵ sec⁻¹ or greater (this dissociation rate constant is theconstant characterizing the dissociation reaction of the complex formedbetween the binding site B of the receptor binding reagent and thereceptor molecule on the surface of the target cell). The associationrate constant (k_(on)) for the association reaction between the bindingsite B of the receptor binding reagent and the receptor molecule on thesurface of the target cell may have any value. In order to ensure asufficiently reversible binding between receptor molecule and receptorbinding reagent it is advantageous to select the k_(off) value of thebinding equilibrium to have a value of about 3×10⁻⁵ sec⁻¹ or greater, ofabout 5×10⁻⁵ sec⁻¹ or greater, such as about 1×10⁻⁴ sec⁻¹ or greater,about 1.5×10⁻⁴ sec⁻¹ or greater, about 2.0×10⁻⁴ sec⁻¹ or greater, about2.5×10⁻⁴ sec⁻¹ or greater, about 3×10⁻⁴ sec⁻¹ or greater, about 3.5×10⁻⁴sec⁻¹ or greater, about 4×10⁻⁴ sec⁻¹ of greater, about 5×10⁻⁴ sec⁻¹ orgreater, about 7.5×10⁻⁴ sec⁻¹ or greater, about 1×10⁻³ sec⁻¹ or greater,about 1.5×10⁻³ sec⁻¹ or greater, about 2×10⁻³ sec⁻¹ or greater, about2.5×10⁻³ sec⁻¹ or greater, about 3×10⁻³ sec⁻¹ or greater, about 4×10⁻³sec⁻¹, about 5×10⁻³ sec⁻¹ or greater, about 7.5×10⁻³ sec⁻¹ or greater,about 1×10⁻² sec⁻¹ or greater, about 5×10⁻² sec⁻¹ or greater, about1×10⁻¹ sec⁻¹ or greater or about 5×10⁻¹ sec⁻¹ or greater. The term“about” when used herein in relation to the k_(off) rate, the k_(on)rate or the K_(D) (see below) is meant to include an error margin of±20.0%, including ±15.0%, ±10.0%, ±8.0%, ±9.0%, ±7.0%, ±6.0%, ±5.0%,±4.5%, ±4.0.%, 3.5%, ±3.0%, ±2.8%, ±2.6%, ±2,4,%, ±2.2%, ±2.0%, ±1.8,%,±1.6%, ±1.4%, ±1.2%, ±1.0, %, ±0.9%, ±0.8%, ±0.7%, ±0.6%, 0.5%, ±0.4%,±0.3%, ±0.2%, ±0.1%, or ±0.01%. It is noted here that the values of thekinetic and thermodynamic constants as used herein, refer to conditionsof atmospheric pressure, i.e. 1.013 bar, and room temperature, i.e. 25°C.

If the receptor binding reagent is symbolized by “A”, the receptor onthe surface of the target cell is symbolized by “B”, and a complexbetween the receptor binding reagent and the receptor is symbolized by“AB”, a bimolecular interaction between the receptor binding reagent andreceptor can be described by a two-state process noted

The corresponding dissociation K_(d) constant of the process is definedas

$K_{d} = {\frac{\lbrack A\rbrack \cdot \lbrack B\rbrack}{\lbrack{AB}\rbrack}.}$

In these equations [A], [B], and [AB] are the equilibrium molarconcentrations of the receptor, the receptor binding reagent (ligand)and the respective complex at a given temperature and a given pressure.The dissociation K_(d) constant can also be expressed as the ratio ofthe constant of the on-rate (k_(on)) for the speed ofassociation/formation, also called association rate constant, of thecomplex and the constant of the off-rate (k_(off)) for the dissociationof the complex, also called dissociation rate constant, with

K _(d) =k _(off) /k _(on)

It is noted in this regard that the dissociation constant K_(d) definesa state where an equilibrium has been reached. No equilibrium may,however, be formed under conditions of chromatographic separation. Thismay explain why in some embodiments it is the constant of the off-rate(k_(off)) rather than the dissociation constant K_(d) that may determinethe reversible binding might be equal to or greater than—i.e. innumerative terms (sec⁻¹) to be at least as high as—3×10⁻⁵ sec⁻¹ in thecontext of the invention.

In some embodiments the receptor binding reagent has a single(monovalent) binding site B capable of specifically binding to thereceptor molecule. In some embodiments the receptor binding reagent hasat least two (i.e., a plurality of binding sites B including three, fouror also five identical binding sites B), capable of binding to thereceptor molecule. In any of these embodiment the binding of thereceptor molecule via (each of) the binding site(s) B may have a k_(off)value of about 3×10⁻⁵ sec⁻¹ or greater. Thus, the receptor bindingreagent can be monovalent (for example a monovalent antibody fragment ora monovalent artificial binding molecule (proteinaceous or other) suchas a mutein based on a polypeptide of the lipocalin family (also knownas “Anticalin®), or a bivalent molecule such as an antibody or afragment in which both binding sites are retained such as an F(ab′)₂fragment. In some embodiments the receptor molecule may be a multivalentmolecule such as a pentameric IgE molecule, provided the k_(off) rate is3×10⁻⁵ sec⁻¹ or greater.

In some embodiments of the invention, it is on a molecular level not thek_(off) rate (of 3×10⁻⁵ sec⁻¹ or greater) of the binding of the receptorbinding reagent via the at least binding site B and the receptormolecule on the target cell that provides for the (traceless) isolationof biological material via reversible cell affinity chromatographytechnology described here. Rather, and as described, for example, inU.S. Pat. No. 7,776,562 or International Patent application WO02/054065,a low affinity binding between the receptor molecule and the bindingsite B of the binding receptor binding reagent together with an avidityeffect mediated via the immobilized affinity reagent allows for areversibly and traceless isolation of a target cell. In theseembodiments a complex between the two or more binding sites Z of theaffinity reagent and the binding partner C of at least two receptorbinding reagents can form, allowing a reversible immobilization andsubsequent elution of the target cells from the affinity chromatographymatrix (via addition of the competing agent that will disrupt thebinding (complex) formed between the binding partner C and the bindingsites Z which in turn leads to the dissociation of the receptor bindingreagent from the target cell. As mentioned above, such a low bindingaffinity may be characterized by a dissociation constant (K_(D)) in therange from about 1.0×10⁻³ M to about 1.0×10⁻⁷ M for the binding of thereceptor binding reagent via the binding site B and the receptormolecule on the target cell surface.

A method according to the present invention may in some embodiments beused to deplete a sample of reagents that have previously been used incell separation. The receptor binding reagent and a competition agentmay, for instance, be present included in the eluate of an affinitychromatography method in a selection cartridge as described above. Usinga method according to the invention such reagents may be at leastessentially, including entirely removed from a sample, e.g. from a cellpopulation. As an illustrative example, a receptor binding reagent asdefined above may be depleted from a sample to levels that are below thedetection limit of e.g. FACS or Western Blot. A competition reagent mayhave been used in order to elute the target cell from an affinitypurification medium such as an affinity chromatography bead. Thiscompetition reagent has a binding site that is capable of specificallybinding to the binding site Z of the affinity reagent. In such anembodiment the respective method of the invention may serve in depletingthe receptor binding reagent and the competition reagent, includingremoving the same.

In some embodiments a method of isolating a target cell may include twopurification steps, of which only the second step, namely the removal ofa receptor binding reagent and/or an competition reagent in a “removalcartridge” is carried out according to the invention. The first stepmight be method of isolating a target cell as described in U.S. Pat.Nos. 7,776,562, 8,298,782 or International Patent application WO02/054065. On such sample a “removal method” according to the presentinvention may then be carried out, to deplete the target cell samplefurther of other cells and also of a receptor binding reagent andcompetition reagent. Likewise, a sample obtained in a first step inaccordance with U.S. Pat. Nos. 7,776,562, 8,298,782 or InternationalPatent application WO 02/054065 can also be subjected to gel permeationchromatography as explained above in which an unmodified chromatographymatrix that does not have an affinity reagent immobilized thereon isused. It is also possible that the first isolation step is any otherknown prior art method for isolation cells, for example, a method thatis described in Example 11 of U.S. Pat. No. 6,022,951, which is thensubjected to a purification method as carried out in the removalcartridge of the present invention.

The receptor molecule that is located on the target cell surface (or anaccessible surface of a biological entity) may be any molecule as longas it remains covalently or non-covalently bonded to the cell surfaceduring a chromatographic separation process in a method according to theinvention. The receptor molecule is a molecule against which a receptorbinding reagent may be directed. In some embodiments the receptor is apeptide or a protein, such as a membrane receptor protein. In someembodiments the receptor is a lipid, a polysaccharide or a nucleic acid.A receptor that is a protein may be a peripheral membrane protein or anintegral membrane protein. It may in some embodiments have one or moredomains that span the membrane. As a few illustrative examples, amembrane protein with a transmembrane domain may be a G-protein coupledreceptor, such as an odorant receptors, a rhodopsin receptor, arhodopsin pheromone receptor, a peptide hormone receptor, a tastereceptor, a GABA receptor, an opiate receptor, a serotonin receptor, aCa²⁺ receptor, melanopsin, a neurotransmitter receptor, such as a ligandgated, a voltage gated or a mechanically gated receptor, including theacetylcholine, the nicotinic, the adrenergic, the norepinephrine, thecatecholamines, the L-DOPA-, a dopamine and serotonin (biogenic amine,endorphin/enkephalin) neuropeptide receptor, a receptor kinase such asserin/threonin kinase, a tyrosine kinase, a porin/channel such as achloride channel, a potassium channel, a sodium channel, an OMP protein,an ABC transporter (ATP-Binding Cassette-Transporter) such as amino acidtransporter, the Na-glucose transporter, the Na⁺/iodide transporter, anion transporter such as Light Harvesting Complex, cytochrome c oxidase,ATPase Na/K, H/K, Ca, a cell adhesion receptor such as metallo protease,an integrin or a catherin.

In some embodiments the receptor molecule may be an antigen defining adesired cell population or subpopulation, for instance a population orsubpopulation of blood cells, e. g. lymphocytes (e.g. T cells, T-helpercells, for example, CD4⁺ T-helper cells, B cells or natural killercells), monocytes, or stem cells, e. g. CD34-positive peripheral stemcells or Nanog or Oct-4 expressing stem cells. Examples of T-cellsinclude cells such as CMV-specific CD8⁺ T-lymphocytes, cytotoxicT-cells, memory T-cells and regulatory T-cells (Treg). An illustrativeexample of Treg are CD4⁺CD25⁺CD45RA Treg cells and an illustrativeexample of memory T-cells are CD62L⁺CD8+ specific central memoryT-cells. The receptor may also be a marker for a tumour cell.

As indicated above, the receptor binding reagent has, in addition to thebinding site B that is able to bind the receptor molecule, a bindingpartner C. This binding partner C is able to bind to a binding site Z ofthe affinity reagent, wherein the multimerization reagent has one ormore binding sites for the binding partner C. The non-covalent bond thatis formed between the binding partner C that is included in the receptorbinding reagent and the binding site(s) Z of the affinity reagent may beof any desired strength and affinity, as long as it is disruptable orreversible under the conditions under which the method of the inventionis performed. The dissociation constant (K_(D)) of the binding betweenthe binding partner C that is included in the receptor binding reagentand the binding site Z of the affinity reagent may have a value in therange from about 10⁻² M to about 10⁻¹³ M. Thus, this reversible bondcan, for example, have a K_(D) from about 10⁻² M to about 10⁻¹³ M, orfrom about 10⁻³ M to about 10⁻¹² M or from about 10⁻⁴ M to about 10⁻¹¹M, or from about 10⁻⁵ M to about 10⁻¹° M. The K_(D) of this bond as wellas the K_(D), k_(off) and k_(m), rate of the bond formed between thebinding site B of the receptor binding reagent and the receptor moleculecan be determined by any suitable means, for example, by fluorescencetitration, equilibrium dialysis or surface plasmon resonance. Thereceptor molecule binding reagent may include at least one, includingtwo, three or more, second binding partners C and the affinity reagentmay include at least two, such as three, four, five, six, seven, eightor more binding sites for the binding partner that is included in thereceptor molecule binding reagent. As described in U.S. Pat. Nos.7,776,562, 8,298,782 or International Patent application WO 2002/054065any combination of a binding partner C and an affinity agent with one ormore corresponding binding sites Z can be chosen, as long as the bindingpartner C and the binding site Z of the affinity agent are able toreversibly bind or multimerize in a (multivalent) complex to cause anavidity effect.

The binding partner included in the receptor binding reagent may forinstance be hydrocarbon-based (including polymeric) and includenitrogen-, phosphorus-, sulphur-, carben-, halogen- or pseudohalogengroups. It may be an alcohol, an organic acid, an inorganic acid, anamine, a phosphine, a thiol, a disulfide, an alkane, an amino acid, apeptide, an oligopeptide, a polypeptide, a protein, a nucleic acid, alipid, a saccharide, an oligosaccharide, or a polysaccharide. As furtherexamples, it may also be a cation, an anion, a polycation, a polyanion,a polycation, an electrolyte, a polyelectrolyte, a carbon nanotube orcarbon nanofoam. Generally, such a binding partner has a higher affinityto the binding site of the multimerization reagent than to other matter.Examples of a respective binding partner include, but are not limitedto, a crown ether, an immunoglobulin, a fragment thereof and aproteinaceous binding molecule with antibody-like functions.

In some embodiments the binding partner C that is included in thereceptor binding reagent includes biotin and the affinity reagentincludes a streptavidin analog or an avidin analog that reversibly bindsto biotin. In some embodiments the binding partner C that is included inthe receptor binding reagent includes a biotin analog that reversiblybinds to streptavidin or avidin, and the affinity reagent includesstreptavidin, avidin, a streptavidin analog or an avidin analog thatreversibly binds to the respective biotin analog. In some embodimentsthe binding partner C that is included in the receptor binding reagentincludes a streptavidin or avidin binding peptide and the affinityreagent includes streptavidin, avidin, a streptavidin analog or anavidin analog that reversibly binds to the respective streptavidin oravidin binding peptide.

In some embodiments the binding partner that is included in the receptorbinding reagent may include a streptavidin-binding peptideTrp-Ser-His-Pro-Gln-Phe-Glu-Lys and the affinity reagent may include thestreptavidin mutein (analog) Val44-Thr45-Ala46-Arg47 or the streptavidinmutein (analog) Ile44-Gly45-Ala46-Arg47, both of which are described inU.S. Pat. No. 6,103,493, for example, and are commercially availableunder the trademark Strep-Tactin®. The streptavidin binding peptidesmight, for example, be single peptides such as the “Strep-Tag®”described in U.S. Pat. No. 5,506,121, for example, or streptavidinbinding peptides having a sequential arrangement of two or moreindividual binding modules as described in International PatentPublication WO 02/077018 or U.S. Pat. No. 7,981,632.

In some embodiment the binding partner C of the receptor binding reagentincludes a moiety known to the skilled artisan as an affinity tag. Insuch an embodiment the affinity reagent includes a corresponding bindingpartner, for example, an antibody or an antibody fragment, known to bindto the affinity tag. As a few illustrative examples of known affinitytags, the binding partner that is included in the receptor bindingreagent may include dinitrophenol or digoxigenin, oligohistidine,polyhistidine, an immunoglobulin domain, maltose-binding protein,glutathione-S-transferase (GST), chitin binding protein (CBP) orthioredoxin, calmodulin binding peptide (CBP), FLAG′-peptide, the HA-tag(sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala), the VSV-G-tag(sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys), the HSV-tag(sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp), the T7 epitope(Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly), maltose binding protein(MBP), the HSV epitope of the sequenceGln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of herpes simplex virusglycoprotein D, the “myc” epitope of the transcription factor c-myc ofthe sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, the V5-tag(sequence: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr), orglutathione-S-transferase (GST). In such an embodiment the complexformed between the one or more binding sites of the affinity reagent, inthis case an antibody or antibody fragment, and the antigen can bedisrupted competitively by adding the free antigen, i.e. the freepeptide (epitope tag) or the free protein (such as MBP or CBP). Theaffinity tag might also be an oligonucleotide tag. Such anoligonucleotide tag may, for instance, be used to hybridize to anoligonucleotide with a complementary sequence, linked to or included inthe affinity reagent.

Further examples of a suitable binding partner include, but are notlimited to, a lectin, protein A, protein G, a metal, a metal ion,nitrilo triacetic acid derivates (NTA), RGD-motifs, a dextrane,polyethyleneimine (PEI), a redox polymer, a glycoproteins, an aptamers,a dye, amylose, maltose, cellulose, chitin, glutathione, calmodulin,gelatine, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine,poly U, or oligo-dT. Lectins such as Concavalin A are known to bind topolysaccharides and glycosylated proteins. An illustrative example of adye is a triazine dye such as Cibacron blue F3G-A (CB) or Red HE-3B,which specifically bind NADH-dependent enzymes. Green A binds to CoAproteins, human serum albumin, and dehydrogenases. The dyes7-aminoactinomycin D and 4′,6-diamidino-2-phenylindole bind to DNA.Cations of metals such as Ni, Cd, Zn, Co, or Cu, are typically used tobind affinity tags such as an oligohistidine containing sequence,including the hexahistidine or theHis-Asn-His-Arg-His-Lys-His-Gly-Gly-Gly-Cys tag (MAT tag), andN-methacryloyl-(L)-cysteine methyl ester.

In some embodiments the binding between the binding partner C that isincluded in the receptor binding reagent and one or more binding sitesof the affinity reagent occurs in the presence of a divalent, atrivalent or a tetravalent cation. In this regard in some embodimentsthe affinity/multimerization reagent includes a divalent, a trivalent ora tetravalent cation, typically held, e.g. complexed, by means of asuitable chelator. The binding partner that is included in the receptorbinding reagent may in such an embodiment include a moiety thatincludes, e.g. complexes, a divalent, a trivalent or a tetravalentcation. Examples of a respective metal chelator, include, but are notlimited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA),ethylene glycol tetraacetic acid (EGTA), diethylenetriaminepentaaceticacid (DTPA), N,N-bis(carboxymethyl)glycine (also called nitrilotriaceticacid, NTA), 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid(BAPTA), 2,3-dimercapto-1-propanol (dimercaprol), porphine and heme. Asan example, EDTA forms a complex with most monovalent, divalent,trivalent and tetravalent metal ions, such as e.g. silver (Ag⁺), calcium(Ca²⁺), manganese (Mn²⁺), copper (Cu²⁺), iron (Fe²⁺), cobalt (Co³⁺) andzirconium (Zr⁴⁺), while BAPTA is specific for Ca²⁺. As an illustrativeexample, a standard method used in the art is the formation of a complexbetween an oligohistidine tag and copper (Cu²⁺), nickel (Ni²⁺), cobalt(Co²⁺), or zinc (Zn²⁺) ions, which are presented by means of thechelator nitrilotriacetic acid (NTA).

In some embodiments the binding partner C that is included in thereceptor binding reagent includes a calmodulin binding peptide and theaffinity reagent includes multimeric calmodulin as described in U.S.Pat. No. 5,985,658, for example. In some embodiments the binding partnerC that is included in the receptor binding reagent includes a FLAGpeptide and the affinity reagent includes an antibody that binds to theFLAG peptide, e.g. the FLAG peptide, which binds to the monoclonalantibody 4E11 as described in U.S. Pat. No. 4,851,341. In one embodimentthe binding partner C that is included in the receptor binding reagentincludes an oligohistidine tag and the affinity reagent includes anantibody or a transition metal ion binding the oligohistidine tag. Thedisruption of all these binding complexes may be accomplished by metalion chelation, e.g. calcium chelation, for instance by adding EDTA orEGTA (supra). Calmodulin, antibodies such as 4E11 or chelated metal ionsor free chelators may be multimerized by conventional methods, e.g. bybiotinylation and complexation with streptavidin or avidin or multimersthereof or by the introduction of carboxyl residues into apolysaccharide, e.g. dextran, essentially as described in Noguchi, A, etal. Bioconjugate Chemistry (1992) 3, 132-137 in a first step and linkingcalmodulin or antibodies or chelated metal ions or free chelators viaprimary amino groups to the carboxyl groups in the polysaccharide, e.g.dextran, backbone using conventional carbodiimide chemistry in a secondstep. In such embodiments the binding between the binding partner C thatis included in the receptor binding reagent and the one or more bindingsites Z of the multimerization reagent can be disrupted by metal ionchelation. The metal chelation may, for example, be accomplished byaddition of EGTA or EDTA.

In some embodiments the affinity reagent is an oligomer or a polymer ofstreptavidin or avidin or of any analog of streptavidin or avidin. Thebinding site Z is the natural biotin binding of avidin or streptavidin.The respective oligomer or polymer may be crosslinked by apolysaccharide. In one embodiment oligomers or polymers of streptavidinor of avidin or of analogs of streptavidin or of avidin are prepared bythe introduction of carboxyl residues into a polysaccharide, e. g.dextran, essentially as described in Noguchi, A, et al., BioconjugateChemistry (1992) 3,132-137 in a first step. Then streptavidin or avidinor analogs thereof may be linked via primary amino groups of internallysine residue and/or the free N-terminus to the carboxyl groups in thedextran backbone using conventional carbodiimide chemistry in a secondstep. Nevertheless, cross-linked oligomers or polymers of streptavidinor avidin or of any analog of streptavidin or avidin may also beobtained by crosslinking via bifunctional molecules, serving as alinker, such as glutardialdehyde or by other methods described in theart.

In the method of the invention the one or more binding sites of thereceptor molecule binding reagent, which specifically binds to thereceptor molecule, may for instance be an antibody, a fragment thereofand a proteinaceous binding molecule with antibody-like functions.Examples of (recombinant) antibody fragments are Fab fragments, Fvfragments, single-chain Fv fragments (scFv), a divalent antibodyfragment such as an (Fab)2′-fragment, diabodies, triabodies (Iliades,P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., etal., Journal of Immunological Methods (2007) 318, 88-94) and otherdomain antibodies (Holt, L. J., et al., Trends Biotechnol. (2003), 21,11, 484-490). In some embodiments one or more binding sites of thereceptor molecule binding reagent may be a bivalent proteinaceousartificial binding molecule such as a dimeric lipocalin mutein that isalso known as “duocalin”. In some embodiments the receptor bindingreagent may have a single second binding site, i.e., it may bemonovalent. Examples of monovalent receptor binding reagents include,but are not limited to, a monovalent antibody fragment, a proteinaceousbinding molecule with antibody-like binding properties or an MHCmolecule. Examples of monovalent antibody fragments include, but are notlimited to a Fab fragment, a Fv fragment, and a single-chain Fv fragment(scFv), including a divalent single-chain Fv fragment.

As mentioned above, an example of a proteinaceous binding molecule withantibody-like functions is a mutein based on a polypeptide of thelipocalin family (see for example, WO 03/029462, Beste et al., Proc.Natl. Acad. Sci. U.S.A. (1999) 96, 1898-1903). Lipocalins, such as thebilin binding protein, the human neutrophil gelatinase-associatedlipocalin, human Apolipoprotein D or human tear lipocalin possessnatural ligand-binding sites that can be modified so that they bind agiven target. Further examples of a proteinaceous binding molecule withantibody-like binding properties that can be used as a receptor bindingreagent that specifically binds to the receptor molecule include, butare not limited to, the so-called glubodies (see e.g. internationalpatent application WO 96/23879), proteins based on the ankyrin scaffold(Mosavi, L. K., et al., Protein Science (2004) 13, 6, 1435-1448) orcrystalline scaffold (e.g. international patent application WO 01/04144)the proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187,AdNectins, tetranectins and avimers. Avimers, including multivalentavimer proteins evolved by exon shuffling of a family of human receptordomains, contain so called A-domains that occur as strings of multipledomains in several cell surface receptors (Silverman, J., et al., NatureBiotechnology (2005) 23, 1556-1561). Adnectins, derived from a domain ofhuman fibronectin, contain three loops that can be engineered forimmunoglobulin-like binding to targets (Gill, D. S. & Damle, N. K.,Current Opinion in Biotechnology (2006) 17, 653-658). Tetranectins,derived from the respective human homotrimeric protein, likewise containloop regions in a C-type lectin domain that can be engineered fordesired binding (ibid.). Peptoids, which can act as protein ligands, areoligo(N-alkyl) glycines that differ from peptides in that the side chainis connected to the amide nitrogen rather than the a carbon atom.Peptoids are typically resistant to proteases and other modifyingenzymes and can have a much higher cell permeability than peptides (seee.g. Kwon, Y-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129,1508-1509).

Yet further examples of suitable proteinaceous binding molecules are anEGF-like domain, a Kringle-domain, a fibronectin type I domain, afibronectin type II domain, a fibronectin type III domain, a PAN domain,a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsinInhibitor domain, tendamistat, a Kazal-type serine protease inhibitordomain, a Trefoil (P-type) domain, a von Willebrand factor type Cdomain, an Anaphylatoxin-like domain, a CUB domain, a thyroglobulin typeI repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, aThrombospondin type I domain, an immunoglobulin domain or a animmunoglobulin-like domain (for example, domain antibodies or camelheavy chain antibodies), a C-type lectin domain, a MAM domain, a vonWillebrand factor type A domain, a Somatomedin B domain, a WAP-type fourdisulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain,“Kappabodies” (cf. Ill. et al., Protein Eng (1997) 10, 949-57, a socalled “minibody” (Martin et al., EMBO J (1994) 13, 5303-5309), adiabody (cf. Holliger et al., PNAS USA (1993)90, 6444-6448), a so called“Janusis” (cf. Traunecker et al., EMBO J (1991) 10, 3655-3659, orTraunecker et al., Int J Cancer (1992) Suppl 7, 51-52), a nanobody, amicrobody, an affilin, an affibody, a knottin, ubiquitin, a zinc-fingerprotein, an autofluorescent protein or a leucine-rich repeat protein. Anexample of a nucleic acid molecule with antibody-like functions is anaptamer. An aptamer folds into a defined three-dimensional motif andshows high affinity for a given target structure.

The term “nucleic acid molecule” as used herein refers to any nucleicacid in any possible configuration, such as single stranded, doublestranded or a combination thereof. Nucleic acids include for instanceDNA molecules, RNA molecules, analogues of the DNA or RNA generatedusing nucleotide analogues or using nucleic acid chemistry, lockednucleic acid molecules (LNA), PNA molecules (supra) and tecto-RNAmolecules (e.g. Liu, B., et al., J. Am. Chem. Soc. (2004) 126,4076-4077). A PNA molecule is a synthetic nucleic acid analogue with apseudopeptide backbone in which the phosphodiester backbone present ine.g. DNA or RNA is replaced by repetitive units of short aliphaticmoieties with an amino end and a carboxylic end, forming an amide bondin the oligomer or polymer. An LNA molecule has a modified RNA backbonewith a methylene bridge between C4′ and O2′, which locks the furanosering in a N-type configuration, providing the respective molecule with ahigher duplex stability and nuclease resistance. Unlike a PNA moleculean LNA molecule has a charged backbone. DNA or RNA may be of genomic orsynthetic origin and may be single or double stranded. Such nucleic acidcan be e.g. mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA,a copolymer of DNA and RNA, oligonucleotides, etc. A respective nucleicacid may furthermore contain non-natural nucleotide analogues and/or belinked to an affinity tag or a label.

A method according to the present invention may be carried out at anytemperature at which the viability of the target cell is at leastessentially uncompromised. When reference is made herein to conditionsthat are at least essentially not harmful, not detrimental or at leastessentially not compromising viability, conditions are referred to,under which the percentage of target cells that can be recovered withfull viability, is at least 70%, including at least 75%, at least 80%,at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, atleast 98%, at least 99% or at least 99.5%. In some embodiments a methodaccording to the invention is carried out at a temperature of about 20°C. or below, such as about 14° C. or below, about 9° C. or below orabout 6° C. or below. Depending on the target cell to be isolated asuitable temperature range may for instance be from about 2° C. to about45° C., including from about 2° C. to about 40° C., from about 3° C. toabout 35° C., or from about 4° C. to about 30° C. if an aqueous mediumis used to encompass the target cell. In some embodiments a methodaccording to the invention is carried out at a constant temperaturevalue, or at a selected temperature value ±about 5° C., about 4° C.,±about 3° C., ±about 2° C., ±about 1° C. or ±about 0.5° C. Thetemperature may, for example, be selected to have a value of about 5°C., about 10° C., about 15° C., about 20° C. or about 25° C. In someembodiments the temperature is altered, i.e. increased, decreased orvaried by combinations thereof, during a method according to the presentinvention. The temperature may for example be altered within a range asdefined above, e.g. in the range from about 2° C. to about 40° C. orwithin the range from about 3° C. to about 35° C. The person skilled inthe art is able to empirically determine a suitable temperature, takinginto account the nature of the cells and the isolation conditions. Forexample, temperature insensitive cells such as cancer cells mightisolated at room temperature or even elevated temperature such as 37° C.

The method may also be carried out using a kit of parts, for instancedesigned for performing a method as detailed above. The kit may includea receptor binding reagent as defined above. The kit may for exampleinclude a container filled with the receptor binding reagent, e.g. insolution. The kit may also include a chromatography matrix as definedabove, which may be (pre)packed into a column, such as a cartridge.Associated with such chromatography matrix and/or container(s) there isin some embodiments provided a notice in the form of instructions on howto use the kit to carry out a method according to the present invention.

The invention also provides for the use of streptavidin, a streptavidinmutein (analogue), avidin, an avidin mutein (analogue) or a mixturethereof for isolation of a target cell via chromatography, wherein thechromatography is a gel filtration chromatography. For this purpose,embodiment, streptavidin, a streptavidin mutein, avidin, an avidinmutein or a mixture of any of these, for example, a mixture of bothstreptavidin and a streptavidin mutein, are immobilized as affinityreagent on a stationary phase of a “removal cartridge” as disclosedherein. The term “streptavidin” as used herein includes wild-typestreptavidin, streptavidin muteins and streptavidin-like polypeptides.Likewise, the term “avidin” as used herein includes wild-type avidin aswell as muteins of avidin such as neutravidin, a deglycosylated avidinwith modified arginines that exhibits a more neutral pI and is availableas an alternative to native avidin. Deglycosylated, neutral forms ofavidin include those commercially available forms such as “Extravidin”,available through Sigma-Aldrich, or “NeutrAvidin” available from ThermoScientific or Invitrogen, for example.

Under wild-type streptavidin (wt-streptavidin), the amino acid sequencedisclosed by Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882 isreferred to. Streptavidin muteins are polypeptides which aredistinguished from the sequence of wild-type streptavidin by one or moreamino acid substitutions, deletions or additions and which retain thebinding properties of wt-streptavidin. Streptavidin-like polypeptidesand streptavidin muteins are polypeptides which essentially areimmunologically equivalent to wild-type streptavidin and are inparticular capable of binding biotin, biotin derivative or biotinanalogues with the same or different affinity as wt-streptavidin.Streptavidin-like polypeptides or streptavidin muteins may contain aminoacids which are not part of wild-type streptavidin or they may includeonly a part of wild-type streptavidin. Streptavidin-like polypeptidesare also polypeptides which are not identical to wild-type streptavidin,since the host does not have the enzymes which are required in order totransform the host-produced polypeptide into the structure of wild-typestreptavidin. The term “streptavidin” also includes streptavidintetramers and streptavidin dimers, in particular streptavidinhomotetramers, streptavidin homodimers, streptavidin heterotetramers andstrepavidin heterodimers. Each subunit normally has a binding site forbiotin or biotin analogues or for streptavidin-binding peptides.Examples of streptavidins or streptavidin muteins are mentioned, forexample, in WO 86/02077, DE 19641876 A1, U.S. Pat. No. 6,022,951, WO98/40396 or WO 96/24606.

In a preferred embodiment, streptavidin muteins that are used forisolation of a target cell via chromatography, wherein thechromatography is a gel filtration chromatography are those streptavidinmuteins which are described in U.S. Pat. No. 6,103,493 and also in DE196 41 876.3. These streptavidin muteins have at least one mutationwithin the region of amino acid positions 44 to 53, based on the aminoacid sequence of wild-type streptavidin. Preference is given to muteinsof a minimal streptavidin, which start N-terminally in the region ofamino acids 10 to 16 of wild-type streptavidin and end C-terminally inthe region of amino acids 133 to 142 of wild-type streptavidin. Examplesof such preferred streptavidin muteins have a hydrophobic aliphaticamino acid instead of Glu at position 44, any amino acid at position 45,a hydrophobic aliphatic amino acid at position 46 or/and a basic aminoacid instead of Val at position 47. The streptavidin mutein may be themutein Val44-Thr45-Ala46-Arg47 or the streptavidin mutein (analog)Ile44-Gly45-Ala46-Arg47, both of which are described in U.S. Pat. No.6,103,493, for example, and which are commercially available under thetrademark Strep-Tactin®.

The invention also provides an apparatus for purification of targetcells, wherein the apparatus comprises at least one arrangement of afirst and a second stationary phase for chromatography as explainedabove, that means a chromatography column for selection of cells (aselection cartridge) and a second chromatography column (a removalcartridge) for removal of reagents of the isolation or the staining oftarget cells. Carrying out such a two step isolation procedure yieldstarget cells which can be directly subjected to the next desiredapplication or selection cycle. In contrast to FACS and MACS selections,in the inventive chromatographic selection method no further proceduressuch as washing and centrifugation are necessary between two selectioncycles and the cells are not functionally compromised by bound isolationreagents such as receptor binding reagents or magnetic beads. Therefore,the invention provides for the first time a reliable, simple toconstruct and yet effective apparatus for target cell purification.

In line with the above, an apparatus of the invention claim may comprisea plurality of arrangements of first and second stationary phases(chromatography columns) being fluidly connected in series. Theapparatus may comprise a sample inlet being fluidly connected to thefirst stationary phase of the first arrangement of a first and a secondstationary phases for chromatography. The apparatus may also comprise asample outlet for purified target cells, the sample outlet being fluidlyconnected to the second stationary phase of the last of the at least onearrangement of a first and second stationary phases for chromatography.The apparatus may also comprise a competition reagent container that isfluidly connected to at least one of the first stationary phases of thearrangements of a first and second stationary phases for chromatography.

As one of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, other compositions of matter,means, uses, methods, or steps, presently existing or later to bedeveloped that perform substantially the same function or achievesubstantially the same result as the corresponding exemplary embodimentsdescribed herein may likewise be utilized according to the presentinvention.

Experimental Examples

In the present examples recombinantly produced Fab fragments that aredirected against cell surface markers are used as receptor bindingreagent. The Fab fragments are recombinantly expressed in E. coli orother hosts and contain a streptavidin binding peptide as bindingpartner C. Tetrameric streptavidin or tetrameric streptavidin muteinsprovides the one or more binding site(s) Z. Fab fragments are bound tostreptavidin, which itself is covalently linked to beads. These beadsare used for affinity column chromatography of cell suspensions with asubpopulation of cells having an extracellular protein (receptormolecule) that is able to be bound by the Fab fragments. Unbound cellsare washed away in this “selection cartridge” while bound cells aresubsequently eluted with biotin containing buffer disrupting the bindingof streptavidin mutein with the streptavidin binding peptide of the Fabfragment (acting as receptor binding reagent). As a consequence, Fabfragment with the cell bound thereon are released from the column and,due to the missing avidity effect, Fab fragments dissociate from thetarget cells. The suspension can now be purified from the remaining Fabfragments and biotin by a second column chromatography (the removalcartridge) using another gel (chromatography) matrix with covalentlybound streptavidin whereby cells elute in the void volume and Fabfragments and biotin are quantitatively bound on the chromatographymatrix. The cells can now be exposed to a further cycle of purificationusing a different Fab fragment or any other receptor binding reagent ina similar way. Columns with Fab fragments (selection cartridge) and asubsequent column for Fab and biotin removal (removal cartridge) can becombined in a serial manner by simply arranging the columns linearly oneafter the other. By so doing, an automated cell purification system asshown in FIG. 6A-6B that is devoid of magnetic beads or any manualinterference and that allows for speedy, easy and cost-efficientpurification of target cells can be provided by the present invention.

This procedure allows, for example, the serial purification of T-cellsstarting with CD4+ purification followed by CD25+ purification from theCD8+ fraction resulting in a highly enriched fraction of regulatoryT-cells as shown below. Further cycles of purification using differentFab fragments are of course possible.

Isolation of CD8+ T-Cells from Human Blood (Typical Single StepPurification) Material and Methods

Human blood was used to isolate PCMBs in a standard procedure.

Example 1: Single Step Purification of CD8+ Cells Via ColumnChromatography

Sephadex G50 (Sigma) was used as stationary phase and was covalentlycoupled with Strep-Tactin® (a recombinant streptavidin variant, IBAGmbH, Germany) using the CNBr method. A 50% suspension of Sephadex G50contained covalently coupled 70 microgram Step-Tactin®/ml of the beadsuspension. Strep-Tactin® served as affinity reagent which wasimmobilized to the affinity matrix before addition of receptor bindingreagent and the sample containing the target cells. An CD8+ binding Fabfragment the heavy chain of which was carboxy-terminally fused with asequential arrangement of two streptavidin binding modules(SAWSHPQFEK(GGGS)₂GGSAWSHPQFEK), commercially available (cataloguenumber: 6-8003) from IBA GmbH, Göttingen, Germany) was used as(monovalent) receptor binding reagent, with the streptavidin bindingpeptide serving as binding partner C.

2 ml of the suspension of Sephadex G50 with Strep-Tactin® was incubatedwith 10 microgram of the CD8+ binding Fab fragment for 20 min at 4° C.in order to allow binding of the Fab fragment to the CD8+ target cells.The suspension was then filled in a plastic minicolumn (Mobitec,Göttingen, Germany) with a 90 micrometer frit at the bottom. This columnthus acts as selection cartridge as defined herein. The column wasequilibrated with PBS (phosphate buffered saline) containing 0.5% bovineserum albumin (PBSA buffer) to give a bed volume of 1 ml. 5 millioncells from the PCMBs in 1 ml PBSA were then loaded onto the column inorder to let the sample penetrate into the affinity chromatographymatrix. The column was then washed with 12 ml PBSA. The washing bufferwas collected and centrifuged at 3000 g for 6 minutes to pellet thecells that were washed from the column (pellet 1). Thereafter, 6 ml PBSAthat contained 0.1 millimolar Biotin (Sigma) as competition reagent wasadded to the column in order to elute the CD8+ cells that werereversibly immobilized on the column via the receptor binding andmultimerization agent. The biotin containing fraction was collected andcentrifuged as above to pellet the cells (pellet2).

Pellets from both fractions were resuspended in 1 ml PBSA buffer foranalysis.

Pellet 1 contained about 3.9 million cells, pellet 2 contained 0.7million cells.

FACS analysis (data not shown) showed that the starting material incomparison to pellet 1 was strongly depleted in CD8+ cells (around 70%),pellet 2 shows CD8+ cells in 68% purity. Thus, CD8+ target cells can beisolated via reversible immobilisation/affinity chromatography.

This result was confirmed by the following experiment for enrichment ofCD8+ cells from PBMC the results of which are shown in FIG. 5.Enrichment was performed on two columns both containing Sephadex-50resin coupled with Strep-Tactin® as explained above. The first column(selection cartridge) (diagrams B-D) was loaded with the CD8 binding Fabfragment commercially available from IBA GmbH described above. Thesecond column (diagrams E-G) served as a negative control to thisselection cartridge (and must not be confused with the removal cartridgewhich is the “second column” arranged after the selection cartridge) andwas not loaded with this CD8 binding Fab fragment. Thus, the firstcolumn should show a CD8-Fab-specific enrichment of cells whereas thesecond column (negative control) should not. In order to measure theenrichment the cell population of CD8+ T-cells within the PBMC's wasdetermined before starting the selection procedure (FIG. 5, diagram A,CD8+ T-cells 18.4%, upper right quadrant). After subjecting the PBMCfraction onto the first column flow through of non retarded cells ismeasured (diagram B, CD8+ T-cells 6.3%). 12.1% (18.4%-6.3%) or 66% ofthe CD8+ T-cells were bound to the column. These cells could then bespecifically eluted by addition of biotin buffer disrupting theStrep-tag/Strep-Tactin® interaction and a subsequent washing step(diagram C+D, CD8+ T-cells 47% and 62.2%). In contrast no enrichment ofCD8+ T-cells was seen in the second column (diagram D-E-F-G) since theCD8+ T-cell population in the flow through fraction (diagram E, CD8+T-cells 17.0%) and the elution fractions (diagram F and G) is notsignificantly differing from the one measured before the selectionprocedure (diagram A). The differences of app. 1% of the applied cellsaccount for unspecifically bound cells on the column showing that 95% ofthe subjected PBMC's pass through the column.

Example 2: Removal of Biotin and Fab from C8+ Cells

CD8+ cells were isolated as described above except that cells afterbiotin elution (6 ml buffer) were directly passed through a column ofSuperflow™ Sepharose® beads (bed volume of 6 ml) that had Strep-Tactin®(IBA GmbH, Göttingen, Germany) covalently attached thereto with abinding capacity of 300 nanomol biotin/ml. While the Superflow™Sepharose® beads served as gel permeation matrix for theisolation/enrichment of target cells, the Strep-Tactin® immobilized onthe beads has binding affinity to both the CD8+ Fab fragments that areequipped with the streptavidin binding peptide and for biotin. Thus, theStrep-Tactin® served as affinity/removal reagent for biotin and the CD8+Fab fragments. The eluate (6 ml) of this second column (which acted asremoval cartridge) filled with Superflow beads was collected.

Biotin and Fab fragments were not detectable in the eluate thatcontained the target cells using a biotin assay and Fab fragment assayusing Western blotting (results not shown). A similar experiment wascarried out using FITC labeled biotin (Sigma) and fluorescently labeledCD8+ Fab (IBA GmbH). The fact that also this final eluate contained noFab fragments or biotin was confirmed when measured with a sensitivefluorimeter. Thus, it was found, that whereas biotin and Fab werecompletely removed, the eluate after Superflow™/Strep-Tactin®chromatography contained 95% to 100% of the CD8+ cells, with no obviousloss of cells.

Example 3: Serial Purification in “Linear Flow” Chromatography

Since the final fraction as described in experiment 2 contained nobiotin and Fab (both would interfere with a subsequent purificationprocedure by blocking Fab binding sites on Strep-Tactin®), the purifiedCD8+ cells can go to another cycle of purification using, for example, aCD25+ Fab fragment (or any other receptor molecule that is present onthe surface of the isolated CD8+ target cells). Such a serialpurification of T-cells can for example be carried out using theapparatus depicted in FIG. 6A or FIG. 6B.

Example 4: Purification of Cells by Chromatography on a Planar Matrix(Strep-Tactin® Coated Nitrocellulose Membrane)

In this experiment the (“three-dimensional”) column chromatographymatrix (beads coated with Strep-Tactin®) used for purification of cellsin Examples 1 and 2 was replaced by a Strep-Tactin® coated planarmatrix.

Experimental Procedure:

-   -   1) Non-covalent attachment of Strep-Tactin® to a nitrocellulose        membrane and purification of CD8+ T cells        For non-covalent attachment of Strep-Tactin® on the membrane, a        piece of nitrocellulose membrane (24 cm², Whatman, UK) was put        in a petri dish and incubated with 4 mg Strep-Tactin® (IBA GmbH)        in 10 ml PBS for 10 minutes and then washed 5 times with 20 ml        PBS. Then 5 microgramm CD8+ Fab fragment (catalogue number:        6-8003-005, IBA GmbH, Göttingen, supra) was added in 5 ml PBS        and incubated for 5 min at 4° C. Then 5 million cells (PBMCs) in        FACS buffer (0.5% BSA (w/v) in PBS pH 7.4) were added and        incubated at 4° C. for 10 min. Then the membrane was washed five        times in 10 ml FACS buffer and the wash fractions were collected        for FACS analysis. The membrane was then incubated for 5 min in        10 ml FACS buffer containing 1 mmol biotin. The resulting        fraction was collected for FACS analysis.

The FACS analysis showed that the biotin containing fraction was 99.1%pure with regard to CD8+ T cells. The yield of CD8+ cells was about 1.5%as compared to the starting material. Thus, this experiment shows thattarget cells can be effectively isolated using planar chromatography ina “batch-like” fashion.

Example 5: Single Step Purification of Human CD8+ Cells Via ColumnChromatography with Superflow™ Agarose

3 ml Superflow Strep-Tactin® (300 nanomol biotin binding, IBA GmbH,Göttingen, Germany)) was loaded onto a minicolumn (Mobitec, Göttingen,Germany). The column was equilibrated with buffer (PBS plus 0.5% bovineserum albumin, “FACS buffer”) and then PBMCs from human blood (10million cells in 0.2 ml FACS buffer) that had previously been incubatedwith 12 microgram Fab directed against CD8 (catalogue number: 6-8003,IBA GmbH, Göttingen) were loaded (as said above, the heavy chain of theFab-fragment was carboxy-terminally fused with a sequential arrangementof two streptavidin binding modules SAWSHPQFEK(GGGS)₂GGSAWSHPQFEK). Thiscolumn acts as selection cartridge as defined herein. The column waswashed with 12 ml FACS buffer by gravity flow and then elution wascarried out with 12 ml FACS buffer containing 1 mM biotin.

The wash fraction (12 ml) of the FACS buffer contained only 1.77% CD8+cells in comparison to the starting fraction (A) which contained 7.98%CD8+ cells, thus 77.8% CD8+ cells were retarded on the column. Theelution with biotin containing buffer fraction (B, 12 ml) resulted inapproximately 65% of the bound CD8+ cells with a purity of 98.5%. Alsothis experiment shows that CD8+ target cells can be isolated viareversible immobilisation/affinity chromatography as described hereinusing commercially available chromatography matrices.

Example 6: Single Step Purification of Human CD8+ Cells Via ColumnChromatography

Human CD8+ cells were purified from density gradient (Ficoll) purifiedPBMCs by the use of a column prepared from 500 μl Strep-Tactin®-agarose(cross-linked agarose was obtained from Agarose Beads Technologies,Madrid, Spain with a reduced exclusion size compared to Superflow™Agarose) bead resin functionalized with 10 μg anti-CD8 Fab-fragments(catalogue number: 6-8003, IBA GmbH, Göttingen). For this purpose, theFab fragment carrying the sequential arrangement of the two streptavidinbinding modules SAWSHPQFEK(GGGS)₂GGSAWSHPQFEK at the C-terminus of theheavy chain was loaded (immobilized) onto the Strep-Tactin®-agarosematrix by pumping 1000 μl Fab containing washing buffer (PBS plus 0.5%bovine serum albumin) over the column at a speed of 300 μl/min prior tothe cell purification. For purification of the target cells 1×10⁸freshly prepared PBMCs (in 2 ml washing buffer) were automaticallyloaded on the column at a flow rate of 300 μl/min with a peristalticpump. Unbound (CD8-negative) cells were subsequently removed from thecolumn by repetitive washing cycles (4×) with a total of 7 ml of washingbuffer at a speed of 2 ml/min. Finally, CD8+ target cells were elutedfrom the column by removing the bound cells from the affinity matrix byaddition of 5 ml, 100 μM D-biotin solution (V=600 μl/min) and elutionwith 5 ml washing buffer at 2 ml/min. Obtained CD8-positive and-negative fractions were analyzed by flow-cytometry. The CD8+ targetcells were purified with a yield of 80% and a purity of 88%. Dot plotsof the respective starting-, negative- and positive fractions as well asthe corresponding purity and yield of a representative selection areshown in FIGS. 7A-7C.

Example 7: Single Step Purification of Human CD8+ Cells Via ColumnChromatography from Whole Blood

Human CD8+ cells were purified from whole blood by the use of a columnprepared from 1200 μl Strep-Tactin®-agarose (cross-linked agarose wasobtained from Agarose Beads Technologies Madrid, Spain with a reducedexclusion size compared to Superflow™ agarose) bead resin functionalizedwith 30 μg anti-CD8 Fab-fragment (catalogue number: 6-8003, IBA GmbH,Göttingen). For this purpose, the Fab fragment carrying the sequentialarrangement of the two streptavidin binding modulesSAWSHPQFEK(GGGS)₂GGSAWSHPQFEK at the C-terminus of the heavy chain wereimmobilized on the Strep-Tactin®-agarose matrix by pumping 1500 μl Fabfragment containing washing buffer (PBS plus 0.5% bovine serum albumin)over the column at a speed of 300 μl/min prior to the cell purification.For purification of the target cells 10 ml freshly drawn whole blood(diluted 1:1 with washing buffer) was automatically loaded on the columnat a flow rate of 300 μl/min with a peristaltic pump. Unbound(CD8-negative) cells were subsequently removed from the column byrepetitive washing cycles (4×) with a total of 13 ml of washing bufferat a speed of 2 ml/min. Finally, CD8+ target cells were eluted from thecolumn by removing the bound cells from the affinity matrix by additionof 10 ml, 100 μM D-biotin solution (V=600 μl/min) and elution with 10 mlwashing buffer at 2 ml/min. Obtained CD8-positive and -negativefractions were analyzed by flow-cytometry. The CD8+ target cells werepurified with a yield of 80% and a purity of 88%. Dot plots of therespective starting-, negative- and positive fractions as well as thecorresponding purity and yield of a representative selection are shownin FIGS. 8A-8C.

Example 8: Pipette Based Single Step Purification of Murine CD4+ Cellsfrom Splenocytes

CD4+ cells were isolated from splenocytes by the use of a pipette tiploaded with 80 μl Strep-Tactin®-agarose bead resin (cross-linked agarosewas obtained from Agarose Beads Technologies, Madrid, Spain with areduced exclusion size compared to Superflow™ agarose) functionalizedwith 2 μg anti-CD4 Fab-fragment. The pipette tips were filled with theagarose material by Phynexus Inc., USA. The Fab fragment used comprisedthe wild-type variable domains of the CD4 binding antibody GK1.5(Dialynas D P et al., Immunol Rev. 1983; 74:29-56, GenBank Entry kappalight chain: M84148.1 GenBank Entry heavy chain: M84149.1) carrying thesequential arrangement of the two streptavidin binding modulesSAWSHPQFEK(GGGS)₂GGSAWSHPQFEK at the C-terminus of the heavy chain).Loading/immobilizing the Fab fragment onto the StrepTactin®-agarosematrix was achieved by pipetting with a handheld electric pipette 400 μlFab fragment containing washing buffer (PBS plus 0.5% bovine serumalbumin) at a speed of 300 μl/min onto the agarose chromatography matrixprior to the cell purification. For purification of the target cells1×10⁷ murine splenocytes (in 0.5 ml washing buffer) were applied ontothe chromatography matrix present in the tip by 3× repeated up-and-downcycles of the sample using a pipette at a speed of 300 μl/min. This“batch like” chromatography procedure with an up- and down movement ofthe buffer containing the cells is equivalent to using a flow-basedmethod for immobilizing the cells on the chromatography matrix. Unbound(CD4-negative) cells were subsequently removed from the tip by triplerepetitive washing (by pipetting the wash buffer up and down) with 1 mlwashing buffer at a speed of 2 ml/min. Finally, CD4+ target cells wereeluted from the tip by removing the bound cells from the affinity matrixby addition of 1 ml, 100 μM D-biotin solution (V=600 μl/min) and elutionwith 2 ml (2×1 ml) washing buffer at a flow rate 2 ml/min. ObtainedCD4-positive and -negative fractions were analyzed by flow-cytometry.The CD8+ target cells were purified with a yield of 95% and a purity of85%. Dot plots of the respective starting-, negative- and positivefractions as well as the corresponding purity and yield of arepresentative selection are shown in FIGS. 9A-9C.

Example 9: Single Step Purification of Human CD4+ Cells Via ColumnChromatography

Human CD4+ cells were isolated from density gradient (Ficoll) purifiedPBMCs by the use of a pipette tip loaded with 80 μlStrep-Tactin®-agarose (cross-linked agarose was obtained from AgaroseBeads Technologies, Madrid, Spain with a reduced exclusion size comparedto Superflow™ Agarose) bead resin functionalized with 2 μg anti-CD4Fab-fragment. The CD4 Fab fragment used was a mutant of the 13B8.2 Fabfragment described in U.S. Pat. No. 7,482,000 and Bes, C., et al. J BiolChem 278, 14265-14273 (2003)). The mutant Fab fragment termed “m13B8.2”carries the variable domain of the CD4 binding murine antibody 13B8.2and a constant domain consisting of constant human CH1 domain of typegamma1 for the heavy chain and the constant human light chain domain oftype kappa, as described in U.S. Pat. No. 7,482,000. Compared tovariable domains of the 13B8.2 Fab fragment in m13B8.2 the His residueat position 91 of the light chain (position 93 in SEQ ID NO: 2) ismutated to Ala and the Arg residue at position 53 of the heavy chain(position 55 in SEQ ID NO: 1) is mutated to Ala. In addition, the Fabfragment m13B8.2 carries a sequential arrangement of the twostreptavidin binding modules SAWSHPQFEK(GGGS)₂GGSAWSHPQFEK at theC-terminus of the heavy chain. The Fab fragment was immobilized on theStrep-Tactin®-agarose matrix with a handheld electric pipette bypipetting 200 μl Fab fragment containing washing buffer at a speed of300 μl/min prior to the cell purification. For selection of the targetcells 1×10⁷ freshly prepared PBMCs (in 0.5 ml washing buffer) (PBS plus0.5% bovine serum albumin) were automatically applied onto thechromatography matrix present in the tip by 3× repeated up-and-downcycles of the sample using a pipette at a speed of 300 μl/min. Unbound(CD4-negative) cells were subsequently removed from the tip by triplerepetitive washing (by pipetting the wash buffer up and down) with 1 mlwashing buffer at a speed of 2 ml/min. Finally, CD4+ target cells wereeluted from the tip by removing bound cells from the affinity matrix byaddition of 1 ml, 100 μM D-biotin solution (V=600 μl/min) and elutionwith 2 ml (2×1 ml) washing buffer at a flow rate of 2 ml/min. ObtainedCD4-positive and -negative fractions were analyzed by flow-cytometry.The CD4+ target cells were purified with a yield of 90% and a purity of99%. Dot plots of the respective starting-, negative- and positivefractions as well as the corresponding purity and yield of arepresentative selection are shown in FIGS. 10 A-10C.

Example 10: Pipette Based Single Step Purification of Human CD4+ Cellsfrom Whole Blood

CD4+ cells were isolated from whole blood by the use of a pipette tiploaded with 80 μl Strep-Tactin®-agarose (cross-linked agarose wasobtained from Agarose Beads Technologies, Madrid, Spain with a reducedexclusion size compared to Superflow™ Agarose) bead resin functionalizedwith 0.5 μg anti-CD4 Fab-fragment. The CD4 binding Fab fragment m13B8.2used in Example 9 was also in Example 10. The Fab fragment wasimmobilized on the Strep-Tactin®-agarose matrix with a handheld electricpipette by pipetting 200 μl Fab containing washing buffer at a speed of300 μl/min prior to the cell isolation. For isolation of the targetcells 2 ml freshly drawn whole blood (diluted 1:1 with washing buffer)(PBS plus 0.5% bovine serum albumin) was automatically applied onto thechromatography matrix present in the tip by 3× repeated up-and-downcycles using a pipette at a speed of 300 μl/min. Unbound (CD4-negative)cells were subsequently removed from the tip by five times repetitivewashing (by pipetting up and down) with 1 ml washing buffer at a speedof 2 ml/min. Finally, CD4+ target cells were eluted from the tip byremoving bound cells from the affinity matrix by addition of 1 ml, 100μM D-biotin solution (V=600 μl/min) and elution with 2 ml (2×1 ml)washing buffer at 2 ml/min. Obtained CD4-positive and -negativefractions were analyzed by flow-cytometry. The CD4+ target cells werepurified with a yield of 88% and a purity of 70%. Dot plots of therespective starting-, negative- and positive fractions as well as thecorresponding purity and yield of a representative selection are shownin FIGS. 11 A-11C.

In this context it is noted that further purification or further use ofthe target cells as obtained in Examples 4 to 11, biotin as the eluentand the Fab fragment as the respective receptor binding reagent can beremoved from the target cell sample by means of the “removal cartridge”as described in Example 2.

The listing or discussion of a previously published document in thisspecification should not necessarily be taken as an acknowledgement thatthe document is part of the state of the art or is common generalknowledge.

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by exemplary embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1-77. (canceled)
 78. A method of isolating a target cell, wherein thetarget cell has a receptor molecule on the target cell surface, themethod comprising: contacting a sample comprising one or more targetcells with a receptor binding reagent, the receptor binding reagentcomprising a binding site B capable of binding to a receptor molecule onthe target cell surface and a binding partner C that comprises astreptavidin-binding peptide, wherein the binding partner C is capableof reversibly binding to a binding site Z of a streptavidin muteinaffinity reagent; and exposing the sample to column chromatography on astationary phase, the stationary phase having the affinity reagentimmobilized thereon, wherein the binding site Z has a lower dissociationconstant (K_(D)) for biotin or a biotin analogue than for the bindingsite C, thereby reversibly immobilizing the one or more target cells onthe stationary phase.
 79. The method of claim 78, wherein the receptorbinding reagent is immobilized on the stationary phase via the affinityreagent prior to applying the sample comprising the target cell to thestationary phase.
 80. A method of isolating a target cell, wherein thetarget cell has a receptor molecule on the target cell surface, themethod comprising exposing a sample comprising one or more target cellsto column chromatography on a stationary phase, said stationary phasehaving a streptavidin mutein affinity reagent immobilized thereto,wherein the streptavidin mutein affinity reagent is reversibly bound toa receptor binding reagent, wherein: the receptor binding reagentcomprises a binding site B capable of binding to the receptor moleculeon the target cell surface and a binding partner C that comprises astreptativin-binding peptide, wherein the binding partner C isreversibly bound to a binding site Z of the streptavidin mutein affinityreagent; and the binding site Z has a lower dissociation constant(K_(D)) for biotin or a biotin analogue than for the binding site C,thereby reversibly immobilizing the one or more target cells on thestationary phase.
 81. The method of claim 78, further comprising elutingthe one or more target cells from the stationary phase.
 82. The methodof claim 80, further comprising eluting the one or more target cellsfrom the stationary phase.
 83. The method of claim 78, wherein thebinding site Z and the binding partner C have a dissociation constant(K_(D)) in the range of about 10⁻²M to 10⁻¹³M.
 84. The method of claim78, wherein the affinity reagent comprises two or more binding sites Zcapable of reversibly binding to the binding partner C comprised in thereceptor binding reagent.
 85. The method of claim 78, further comprisingloading onto the stationary phase a competition reagent, wherein thecompetition reagent is able to disrupt the binding of the bindingpartner C of the receptor binding reagent to the binding site Z of theaffinity reagent, thereby displacing the receptor binding agent.
 86. Themethod of claim 85, wherein the competition reagent is biotin or abiotin analogue.
 87. The method of claim 78, wherein the receptorbinding reagent is selected from the group consisting of an antibodyfragment, a proteinaceous binding molecule with immunoglobulin-likefunctions, an aptamer and an MHC molecule.
 88. The method of claim 78,wherein the binding site B of the receptor binding reagent ismonovalent.
 89. The method of claim 87, wherein the antibody fragment isselected from a Fab fragment, an Fv fragment or a single chain Fvfragment.
 90. The method of claim 78, wherein the streptavidin muteincomprises the amino acid sequence Val⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷ at sequencepositions 44 to 47 of wild type streptavidin or the streptavidin muteincomprises the amino acid sequence Ile⁴⁴-Gly⁴⁵-Ala⁴⁶-Arg⁴⁷ at sequencepositions 44 to 47 of wild type streptavidin.
 91. The method of claim90, wherein the streptavidin mutein comprises an N-terminal amino acidresidue starting at an amino acid in the region of amino acids 10 to 16of the wildtype streptavidin amino acid sequence and ending in theregion of amino acids 133 to 142 of the wildtype streptavidin amino acidsequence.
 92. The method of claim 80, wherein the streptavidin muteincomprises the amino acid sequence Val⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷ at sequencepositions 44 to 47 of wild type streptavidin or the streptavidin muteincomprises the amino acid sequence Ile⁴⁴-Gly⁴⁵-Ala⁴⁶-Arg⁴⁷ at sequencepositions 44 to 47 of wild type streptavidin.
 93. The method of claim92, wherein the streptavidin mutein comprises an N-terminal amino acidresidue starting at an amino acid in the region of amino acids 10 to 16of the wildtype streptavidin amino acid sequence and ending in theregion of amino acids 133 to 142 of the wildtype streptavidin amino acidsequence.
 94. The method of claim 78, wherein the streptavidin-bindingpeptide comprises the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ IDNO:3).
 95. The method of claim 94, wherein the streptavidin-bindingpeptide comprises two or more individual binding modules comprising thesequence set forth by SEQ ID NO:3.
 96. The method of claim 95, whereinthe streptavidin-binding peptide comprises the sequenceSAWSHPQFEK(GGGS)₂GGSAWSHPQFEK (SEQ ID NO:13).
 97. The method of claim80, wherein the streptavidin-binding peptide comprises the sequenceTrp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:3).
 98. The method of claim97, wherein the streptavidin-binding peptide comprises two or moreindividual binding modules comprising the sequence set forth by SEQ IDNO:3.
 99. The method of claim 98, wherein the streptavidin-bindingpeptide comprises the sequence SAWSHPQFEK(GGGS)₂GGSAWSHPQFEK (SEQ IDNO:13).
 100. The method of claim 78, wherein the sample comprises a bodyfluid.
 101. The method of claim 100, wherein the body fluid is blood ora blood component.
 102. The method of claim 78, wherein the target cellis a mammalian cell.
 103. The method of claim 78, wherein the targetcell is a leukocyte or a stem cell.
 104. The method of claim 103,wherein the target cell is a leukocyte and the leukocyte is alymphocyte.